WO2021231891A1 - Method for direct amplification and detection of rna - Google Patents
Method for direct amplification and detection of rna Download PDFInfo
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- WO2021231891A1 WO2021231891A1 PCT/US2021/032493 US2021032493W WO2021231891A1 WO 2021231891 A1 WO2021231891 A1 WO 2021231891A1 US 2021032493 W US2021032493 W US 2021032493W WO 2021231891 A1 WO2021231891 A1 WO 2021231891A1
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- rna
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- replicase
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- amplicon
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- C—CHEMISTRY; METALLURGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Definitions
- the subject matter relates to methods for direct amplification of target RNA molecules using an RNA replicase, also known as an RNA-dependent RNA polymerase (RdRP), and detection of the amplified target RNA molecules, for molecular diagnosis of pathogens in body fluid or tissue samples.
- RNA replicase also known as an RNA-dependent RNA polymerase (RdRP)
- RdRP RNA-dependent RNA polymerase
- Compositions and kits for use in the direct amplification and detection of target RNA molecules for molecular diagnosis of pathogens are also described herein.
- PCR-based assays for detection of various pathogens, particularly viruses, bacteria and parasites, in clinical samples offer the advantages of high sensitivity and reproducibility, and can be carried out much more rapidly than traditional culturing methods, which can take multiple days. In many cases, rapid analysis is essential in order to properly treat infected individuals and, if necessary, implement procedures to prevent further transmission of infection.
- Pathogens of significance which are discussed further below, include various eukaryotic, prokaryotic, and viral pathogens, in particular, viral, bacterial and/or parasitic infections.
- PCR-based molecular testing allows for sensitive detection of these and other pathogens in patient specimens, in less time than culture testing. However, most PCR protocols nonetheless employ multiple preparation steps, requires testing facilities to have access to a thermocyler, can be time-consuming, and stringent precautions may be needed to avoid contamination of samples.
- RNA amplification plays an important role in the detection of viral infections in patient material.
- RNA viruses i.e., viral pathogens with a genetic material consisting of RNA.
- RNA amplification can also play an important role in the detection of various eukaryotic and prokaryotic pathogens, in particular, bacterial and/or parasitic infections.
- typically RNA has been detected with PCR- based assays by first generating by first generating a complimentary DNA (cDNA) strand of the target RNA with an RNA-dependent DNA polymerase (RdDP), also known as a reverse transcriptase.
- cDNA complimentary DNA
- RdDP RNA-dependent DNA polymerase
- DdDP DNA-dependent DNA polymerase
- a method for detecting a target RNA in a sample comprises (a) adding the sample to an amplification mixture comprising: (i) an RNA replicase (RNA- dependent RNA polymerase); (ii) at least one target oligonucleotide primer specific for the target RNA; (iii) nucleoside triphosphates (NTPs) ATP, CTP, GTP and UTP; (iv) at least one or more of a detectable, target probe for detection of the target RNA; and (v) optionally, an RNAse inhibitor, one or more of a reaction helper protein, or combinations thereof; (b) producing at least one or more of an RNA amplicon using RNA replicase-dependent amplification (RDA) of the target RNA, if present in the sample; and (c) hybridizing the at least one or more of the target probe to the at least one or more of the RNA amplicon to detect presence or absence of the target RNA
- RDA RNA replicase-dependent
- the amplification mixture further comprises a control oligonucleotide primer pair specific to a process control sequence and a control probe for detection of the process control sequence.
- the at least one target oligonucleotide primer and control oligonucleotide primer pair are designed such that amplification and detection of the target RNA and the process control sequence can be performed simultaneously using the same amplification conditions.
- At least one or more components of the amplification mixture is provided in a form selected from a solid, solution, lyophilized, frozen liquid form, and any combinations thereof.
- the target RNA is selected from a viral RNA, an mRNA, and a non coding RNA.
- the non-coding RNA is selected from rRNA, tRNA, and snRNA.
- the target RNA is a single-stranded RNA (ssRNA), a double- stranded RNA (dsRNA), or combinations thereof.
- ssRNA single-stranded RNA
- dsRNA double- stranded RNA
- the at least one or more of the RNA amplicon is a single-stranded
- RNA amplicon ssRNA amplicon
- dsRNA amplicon double-stranded RNA amplicon
- the at least one or more of the target probe is a single-strand probe that hybridizes to the ssRNA amplicon, one strand of the dsRNA amplicon, or both strands of the dsRNA amplicon.
- the at least one or more of the target probe is an optically-labeled probe.
- the optically-labeled probe comprises a fluorescent compound.
- the amplification is real-time amplification and producing the at least one or more of the RNA amplicon is determined real-time.
- producing the at least one or more of the RNA amplicon is determined by an end-point analysis.
- the end-point analysis is achieved by a lateral-flow device.
- the RDA is performed essentially isothermally.
- the RDA is performed between 20°C and 75°C.
- the RDA is performed between 35°C and 70°C.
- the amplification mixture comprises a single target oligonucleotide primer.
- the amplification mixture comprises a first target oligonucleotide primer and a second target oligonucleotide primer.
- the first and second target oligonucleotide primers are provided at equal concentrations.
- the first and second target oligonucleotide primers are provided at unequal concentrations.
- the first target oligonucleotide primer is provided at a concentration of at least three times greater than the concentration of the second target oligonucleotide primer.
- the second target oligonucleotide primer is provided at concentration within a range of about 25 nM to about 5 mM.
- the target RNA is a double-stranded RNA, and wherein the first and second target oligonucleotide primers hybridize to opposite strands of the double-stranded target RNA in 5’ to 3’ orientation.
- the one or more of the reaction helper protein is a protein that assists in activity of the RNA replicase (RNA-dependent RNA polymerase), stability of the target RNA, separating double-stranded RNA into single-stranded RNA, and combinations thereof.
- RNA replicase RNA-dependent RNA polymerase
- the one or more of the reaction helper protein is an RNA chaperone, an RNA helicase, an RNA binding-factor, a scaffold protein, an RNA modifying enzyme, other types of RNA-binding protein, and combinations thereof.
- the RNA replicase is a recombinant RNA replicase (RNA-dependent RNA polymerase).
- RNA-dependent RNA polymerase RNA-dependent RNA polymerase
- NSV nonstructural protein 5B
- HCV hepatitis C virus
- the recombinant RNA replicase comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
- presence of the at least one or more of the RNA amplicons is diagnostic for a pathogen or a mutation in a genomic RNA.
- the pathogen is a eukaryotic pathogen, a prokaryotic pathogen, or a virus.
- the virus uses cognate RNA replicases (RNA-dependent RNA polymerases) for replication.
- RNA replicases RNA-dependent RNA polymerases
- the virus is selected from a positive-strand genomic RNA virus, a negative-strand genomic RNA virus, and a double-strand genomic RNA virus.
- an in vitro method for detecting an RNA molecule in a sample comprises combining the sample with an amplification composition comprising (i) an RNA replicase (RNA-dependent RNA polymerase); (ii) at least one oligonucleotide primer specific for the RNA molecule; (iii) nucleoside triphosphates (NTPs) ATP, CTP, GTP and UTP; (iv) at least one or more of a probe comprising a detectable moiety; and (v) optionally, an RNAse inhibitor, one or more of a reaction helper protein, or combinations thereof; generating at least one or more of an RNA amplicon via RNA replicase-dependent amplification (RDA) of the RNA molecule, if present in the sample; and inspecting for presence or absence of signal from the detectable moiety to ascertain presence or absence of the RNA molecule.
- an amplification composition comprising (i) an RNA replicase (RNA-dependent RNA polymerase); (
- the at least one or more of the RNA amplicon is generated without formation of a cDNA or DNA.
- the combining forms a solution of the sample and the amplification composition.
- the RNA molecule is selected from a viral RNA molecule, an mRNA molecule, and a non-coding RNA molecule.
- the non-coding RNA molecule is selected from an rRNA, a tRNA, and an snRNA molecule.
- the target RNA is a single-stranded RNA (ssRNA), a double- stranded RNA (dsRNA), or combinations thereof.
- ssRNA single-stranded RNA
- dsRNA double- stranded RNA
- the at least one or more of the RNA amplicon is a single-stranded
- RNA amplicon ssRNA amplicon
- dsRNA amplicon double-stranded RNA amplicon
- the at least one or more of the probe is a single-strand probe that hybridizes to the ssRNA amplicon, one strand of the dsRNA amplicon, or both strands of the dsRNA amplicon.
- the probe is an optically-labeled probe.
- the optically-labeled probe comprises a fluorescent compound.
- the amplification is real-time amplification and producing the at least one or more of the RNA amplicon is determined real-time.
- producing the at least one or more of the RNA amplicon is determined by an end-point analysis.
- the end-point analysis is achieved by a lateral-flow device.
- the RDA is performed essentially isothermally.
- the RDA is performed between 20°C and 75°C or between 30°C and 65°C.
- the amplification composition comprises a single oligonucleotide primer.
- the amplification composition comprises a first oligonucleotide primer and a second oligonucleotide primer.
- the first and second oligonucleotide primers are provided at equal concentrations.
- the first and second oligonucleotide primers are provided at unequal concentrations.
- the first target oligonucleotide primer is provided at a concentration of at least three times greater than the concentration of the second target oligonucleotide primer.
- the second oligonucleotide primer is provided at concentration within a range of about 25 nM to about 5mM.
- the RNA molecule is a double-stranded RNA molecule, and wherein the first and second oligonucleotide primers hybridize to opposite strands of the double- stranded RNA molecule in 5’ to 3’ orientation.
- the one or more of the reaction helper protein is a protein that assists in activity of the RNA replicase (RNA-dependent RNA polymerase), stability of the RNA molecule, separating double-stranded RNA into single-stranded RNA, and combinations thereof.
- RNA replicase RNA-dependent RNA polymerase
- stability of the RNA molecule separating double-stranded RNA into single-stranded RNA, and combinations thereof.
- the one or more of the reaction helper protein is an RNA chaperone, an RNA helicase, an RNA binding-factor, a scaffold protein, an RNA modifying enzyme, other types of RNA-binding protein, and combinations thereof.
- the RNA replicase is a recombinant RNA replicase.
- the recombinant RNA replicase is derived from nonstructural protein 5B (NS5B) of hepatitis C virus (HCV).
- the recombinant RNA replicase comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
- presence of the least one or more of the RNA amplicon is diagnostic for a pathogen or a mutation in a genomic RNA.
- the pathogen is a eukaryotic pathogen, a prokaryotic pathogen, or a virus.
- the virus uses cognate RNA-dependent RNA polymerases for replication.
- the virus is selected from a positive-strand genomic RNA virus, a negative-strand genomic RNA virus, and a double-strand genomic RNA virus.
- a composition for detecting a target RNA in a sample comprises (i) an RNA replicase (RNA-dependent RNA polymerase); (ii) at least one oligonucleotide primer specific for the target RNA; (iii) nucleoside triphosphates (NTPs) ATP, CTP, GTP and UTP; (iv) at least one or more of a detectable, single-strand probe that hybridizes to the target RNA and an RNA amplicon thereof; and (v) optionally, an RNAse inhibitor, one or more of a reaction helper protein, or combinations thereof.
- RNA replicase RNA-dependent RNA polymerase
- NTPs nucleoside triphosphates
- composition when combined with a sample is capable of producing a single-stranded RNA amplicon (ssRNA amplicon), a double-stranded RNA amplicon (dsRNA amplicon), or combinations thereof, using an isothermal RNA replicase-dependent amplification (RDA) of the target RNA, if present in the sample.
- ssRNA amplicon single-stranded RNA amplicon
- dsRNA amplicon double-stranded RNA amplicon
- RDA isothermal RNA replicase-dependent amplification
- the RNA replicase is a recombinant RNA replicase derived from nonstructural protein 5B (NS5B) of hepatitis C virus (HCV).
- NS5B nonstructural protein 5B
- HCV hepatitis C virus
- the recombinant RNA replicase comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
- kits comprising a first container containing a composition as described herein is provided.
- the kit comprises a second container comprising a master mix, a third container comprising a process buffer, a fourth container containing process control, or any combinations thereof.
- compositions of one or more containers are provided in lyophilized form, frozen liquid form, or combinations thereof.
- the kit further comprises a fifth container containing a rehydration solution.
- the rehydration solution comprises one or more multivalent cation cofactors for catalyzing enzymes and one or more components for maintaining an optimal pH buffer for an efficient amplification reaction.
- RNA replicase comprising the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto is provided.
- the recombinant RNA replicase comprises a polypeptide tag.
- the polypeptide tag is a hexa-histidine peptide (HIS) tag.
- the recombinant RNA replicase comprises a deletion in the transmembrane domain.
- nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 4, or an nulciec acid sequence having at least 80%, 90%, or 95% sequence identity thereto is provided
- plasmid or vector comprising a nucleic acid disclosed herein is provided.
- a host cell comprising disclosed herein is provided.
- a method of generating the recombinant RNA replicase disclosed herein comprising (i) culturing the host cell of claim 80 under conditions sufficient for the expression of the recombinant RNA replicase, and (ii) obtaining the recombinant RNA replicase from the culture.
- exemplary amino acid and nucleic acid sequences, plasmids, vectors, and host cells useful in embodiments of the methods, compositions, and kits described herein. These include those disclosed in the tables herein.
- FIG. 1 provides the amino acid sequence of a wild-type hepatitis C virus (HCV) nonstructural 5B protein (NS5B) RNA-dependent RNA polymerase (RNA replicase), which comprises the amino acid residues 2420 to 3010 of Uniprot P26663, herein identified as SEQ ID NO: 1
- HCV hepatitis C virus
- NS5B nonstructural 5B protein
- RNA replicase RNA-dependent RNA polymerase
- FIG. 2 provides the nucleic acid sequence encoding a wild-type hepatitis C virus
- HCV nonstructural 5B protein
- RNA replicase RNA replicase
- FIG. 3 provides the amino acid sequence of a recombinant hepatitis C virus (HCV) nonstructural 5B protein (NS5B) RNA-dependent RNA polymerase (RNA replicase), herein identified as SEQ ID NO: 3.
- HCV hepatitis C virus
- NS5B nonstructural 5B protein
- RNA replicase RNA-dependent RNA polymerase
- FIG. 4 provides the nucleic acid sequence encoding a recombinant hepatitis C virus
- HCV nonstructural 5B protein
- RNA replicase RNA replicase
- SEQ ID NO: 1 Amino acid sequence of a wild-type hepatitis C virus (HCV) nonstructural 5B protein (NS5B) RNA-dependent RNA polymerase (RNA replicase), which comprises the amino acid residues 2420 to 3010 of Uniprot P26663.
- HCV hepatitis C virus
- NS5B nonstructural 5B protein
- RNA replicase RNA-dependent RNA polymerase
- SEQ ID NO: 2 Nucleic acid (DNA) sequence encoding a wild-type hepatitis C virus
- HCV nonstructural 5B protein
- NS5B RNA-dependent RNA polymerase
- SEQ ID NO: 3 Amino acid sequence of a recombinant HCV NS5B RNA-dependent
- RNA polymerase RNA replicase
- SEQ ID NO: 4 Nucleic acid (DNA) sequence encoding a recombinant HCV NS5B
- RNA-dependent RNA polymerase RNA replicase
- This specification describes various exemplary embodiments of methods, compositions, kits, amino acid and nucleic acid sequences, plasmids, vectors, host cells and the like related to direct amplification of target RNA molecules using an RNA replicase, also known as an RNA-dependent RNA polymerase (RdRP), and detection of the amplified target RNA molecules with various embodiments within the disclosure, for molecular diagnosis of pathogens in body fluid or tissue samples.
- an RNA replicase also known as an RNA-dependent RNA polymerase (RdRP)
- RdRP RNA-dependent RNA polymerase
- compositions, kits, amino acid and nucleic acid sequences, plasmids, vectors, host cells and the like are described, it will be understood that this disclosure is not limited to particular embodiments described, and as such may vary. A number of various embodiments of the present disclosure are described in detail hereinafter. These embodiments may take many different forms and should not be construed as limited to those embodiments explicitly set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present disclosure to those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
- the term “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
- a process, method, compound, composition, kit, an amino acid sequence, a nucleic acid sequence, a plasmid, a vector, a host cell and the like that comprises a list of features is not necessarily limited only to those features but may include other features not expressly listed or inherent to such process, method, compound, composition, kit, amino acid sequence, nucleic acid sequence, plasmid, vector, host cell and the like.
- use of the term “about” indicates that a particular value is intended to include a range of values that represents the limits of accuracy of the instrumentation that was used to obtain the value, or within the range of reproducibility of the value that is being measured by a particular instrument.
- oligonucleotide primer e.g., PCR primers or nucleic acid amplification primers
- the primers are substantially complementary, and in some embodiments exactly complementary, to selected primer binding sites in the target DNA or RNA sequence or DNA or RNA sequence of the target pathogen.
- the selected primer binding sites are in highly conserved regions of the genome. The sequences of the highly conserved regions may be consensus sequences from sequence alignment of multiple strains of the pathogen.
- the definition also applies to nucleic acid probes or PCR probes.
- substantially complementary with respect to a PCR primer or probe or a nucleic acid amplification primer or probe, is meant that the oligomer or oligonucleotide primer or probe is sufficiently complementary to its binding site, e.g., in a DNA, cDNA, or an RNA sequence, for efficient binding and amplification to proceed under the conditions of a PCR assay or conditions of other nucleic acid amplification assays, e.g., nucleic acid amplifications conducted under isothermal conditions.
- the oligomer or oligonucleotide primer or probe is exactly complementary to its binding site or to a consensus sequence for the binding site, e.g., in a DNA, cDNA, or an RNA sequence.
- a DNA, cDNA, or RNA sequence there may be one or more mismatches between the primer and/or probe and the binding site in the analyte (e.g., a DNA, cDNA, or RNA sequence) that are tolerated and still result in specific amplification and detection.
- substantially means sufficient to work for the intended purpose.
- the term “substantially” thus allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, or the like such as would be expected by a person of ordinary skill in the field but that do not appreciably affect overall performance.
- “substantially” means within five, ten, or twenty percent.
- detection of a target nucleic acid or analyte refers to determining the presence or the absence of the nucleic acid or analyte in a sample, where absence refers to a zero level or an undetectable level.
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- A adenine
- T thymine
- C cytosine
- G guanine
- Certain pairs of nucleotides specifically bind to one another in a complementary fashion (called complementary base pairing). That is, adenine (A) pairs with thymine (T) (in the case of RNA, however, adenine (A) pairs with uracil (U)), and cytosine (C) pairs with guanine (G).
- polynucleotide As used herein, the terms “polynucleotide,” “nucleic acid,” “oligonucleotide,”
- oligomer refers to molecules that comprises a polymeric arrangement of nucleotide base monomers, where the sequence of monomers defines the polynucleotide.
- Polynucleotides can include polymers of deoxyribonucleotides to produce deoxyribonucleic acid (DNA), and polymers of ribonucleotides to produce ribonucleic acid (RNA).
- a polynucleotide can be single- or double-stranded. When single stranded, the polynucleotide can correspond to the sense or antisense strand of a gene.
- a single-stranded polynucleotide can hybridize with a complementary portion of a target polynucleotide to form a duplex, which can be a homoduplex or a heteroduplex.
- the term "polynucleotide” is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA).
- mRNA messenger RNA
- pDNA plasmid DNA
- a polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
- a polynucleotide comprises at least three nucleosides.
- oligonucleotides range in size from a few monomeric units, e.g., 3-4, to several hundreds of monomeric units.
- a polynucleotide such as an oligonucleotide is represented by a sequence of letters, such as “ATGCCTG,” it will be understood that the nucleotides are in 5'->3' order from left to right and that “A” denotes deoxy adenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine, unless otherwise noted.
- the letters A, C, G, and T may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art.
- subject or “individual” or “animal” or “patient” or “mammal,” as used herein, generally refers to an animal, such as a mammal (e.g., human) or avian (e.g., bird), or other organism, such as a plant.
- the subject can be a vertebrate, a mammal, a rodent (e.g., a mouse), a primate, a simian, or a human.
- Animals may include, but are not limited to, farm animals, sport animals, and pets.
- a subject can be a healthy or asymptomatic individual, an individual that has or is suspected of having a disease (e.g., cancer) or a pre-disposition to the disease, and/or an individual that is in need of therapy or suspected of needing therapy.
- the term subject includes any subject, particularly a mammalian subject, e.g., a human patient, for whom diagnosis, prognosis, prevention, or therapy is desired.
- An "isolated" polynucleotide is a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding aa protein, e.g., RNA replicase, contained in a vector is considered isolated.
- an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
- Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides described herein. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically.
- polynucleotides may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
- polynucleotide As used herein, it is not intended that the term “polynucleotide” be limited to naturally occurring polynucleotide structures, naturally occurring nucleotides sequences, naturally occurring backbones or naturally occurring intemucleotide linkages.
- polynucleotide analogues One familiar with the art knows well the wide variety of polynucleotide analogues, unnatural nucleotides, non-natural phosphodiester bond linkages and intemucleotide analogs that find use with the compositions and methods described herein.
- nucleic acid sequence refers to the order of nucleotide monomers in the nucleotide polymer. By convention, a nucleotide sequence is typically written in the 5’ to 3’ direction. Unless otherwise indicated, any particular polynucleotide sequence described herein optionally encompasses complementary sequences, in addition to the sequence explicitly indicated.
- sample generally refers to a “biological sample” of a subject.
- the sample can refer to a test sample or a clinical sample, which can include any suitable bodily sample.
- the sample may be obtained from a tissue or body fluid of a subject.
- the body fluid can be any of urine, blood, plasma, serum, or any other suitable body fluid.
- a body fluid or biological fluid can be a solid, or semi-solid sample, including feces, biopsy specimens, skin, nails, and hair, or a liquid sample, such as urine, saliva, sputum, mucous, blood, blood components such as plasma or serum, amniotic fluid, semen, vaginal secretions, tears, spinal fluid, washings, and other bodily fluids.
- the sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate.
- the sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample.
- the sample may be a skin sample.
- the sample may be a cheek swab.
- the sample may be a plasma or serum sample.
- swab specimens from, e.g., the cervix, urethra, nostril, and throat. Any of such samples may be from a living, dead, or dying animal or a plant. Animals include mammals, such as humans.
- the sample may be a cell sample.
- a cell may be a live cell.
- the sample may be a cell line or cell culture sample.
- the sample can include one or more cells.
- the sample can include one or more microbes or pathogens.
- the biological sample may be a nucleic acid sample, e.g., DNA or RNA.
- the sample may be derived from another sample.
- the sample may be a cell-free or cell free sample.
- a cell-free sample may include extracellular polynucleotides, e.g., DNA or RNA.
- Extracellular polynucleotides may be isolated from a bodily sample that may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool, tears, or any other suitable bodily sample.
- sample can refer to a cell or nuclei suspension extracted from a single biological source (blood, tissue, etc.).
- the sample may comprise any number of macromolecules, for example, cellular macromolecules.
- the sample maybe or may include one or more constituents of a cell, but may not include other constituents of the cell.
- An example of such cellular constituents is a nucleus or an organelle.
- the sample may be or may include DNA, RNA, organelles, proteins, or any combination thereof.
- the sample may be or include a chromosome or other portion of a genome.
- the sample may be a cell or one or more constituents from a cell, such as DNA, RNA, nucleus, organelles, proteins, or any combination thereof, from the cell.
- amplicon generally refers to a piece of a genetic fragment or nucleic acid sequence, e.g., a DNA or an RNA, which can be the source and/or product of amplification or replication events.
- amplicon refers to the product of an amplification reaction, and hence can be used interchangeably with common laboratory terms, such as a "PCR product.”
- amplification can refer to the production of one or more copies of a genetic fragment or target sequence of DNA or RNA, specifically the amplicon.
- An amplicon can be formed artificially, using various methods including polymerase chain reactions (PCR), other nucleic acid amplification methods, e.g., nucleic acid amplifications conducted under isothermal conditions or essentially isothermal conditions, or ligase chain reactions (LCR), or naturally through gene duplication.
- Various enzymes can generate DNA or RNA amplicons, for example, a DNA polymerase can generate a DNA amplicon, a reverse transcriptase (RT) or RNA-directed DNA polymerase can generate a DNA amplicon (cDNA) of target mRNA molecules, an RNA replicase or RNA-dependent RNA polymerase can generate a RNA amplicon of target RNA molecules, etc.
- the term “gene” generally refers to a combination of polynucleotide elements, that when operatively linked in either a native or recombinant manner, provide some product or function.
- the term “gene” is to be interpreted broadly, and can encompass mRNA, cDNA, cRNA and genomic DNA forms of a gene.
- the term “gene” encompasses the transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region will contain “open reading frames” that encode polypeptides.
- a “gene” comprises only the coding sequences (e.g . , an “open reading frame” or "coding region") necessary for encoding a polypeptide.
- genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes.
- rRNA ribosomal RNA genes
- tRNA transfer RNA
- the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters.
- the term “gene” encompasses mRNA, cDNA and genomic forms of a gene.
- vector As used herein, the terms “vector,” “vehicle,” “construct” and “plasmid” are used in reference to any recombinant polynucleotide molecule that can be propagated and used to transfer nucleic acid segment(s) from one organism to another.
- Vectors generally comprise parts which mediate vector propagation and manipulation (e.g., one or more origin of replication, genes imparting drug or antibiotic resistance, a multiple cloning site, operably linked promoter/enhancer elements which enable the expression of a cloned gene, etc.).
- Vectors are generally recombinant nucleic acid molecules, often derived from bacteriophages, or plant or animal viruses.
- Plasmids and cosmids refer to two such recombinant vectors.
- a “cloning vector” or “shuttle vector” or “subcloning vector” contain operably linked parts that facilitate subcloning steps (e.g., a multiple cloning site containing multiple restriction endonuclease target sequences).
- a nucleic acid vector can be a linear molecule, or in circular form, depending on type of vector or type of application. Some circular nucleic acid vectors can be intentionally linearized prior to delivery into a cell.
- the term “expression vector” refers to a recombinant vector comprising operably linked polynucleotide elements that facilitate and optimize expression of a desired gene (e.g. , a gene that encodes a protein) in a particular host organism (e.g., a bacterial expression vector or mammalian expression vector).
- a desired gene e.g., a gene that encodes a protein
- a particular host organism e.g., a bacterial expression vector or mammalian expression vector.
- Polynucleotide sequences that facilitate gene expression can include, for example, promoters, enhancers, transcription termination sequences, and ribosome binding sites.
- the term “host cell” refers to any cell that contains a heterologous nucleic acid.
- the heterologous nucleic acid can be a vector, such as a shuttle vector or an expression vector.
- the host cell is able to drive the expression of genes that are encoded on the vector.
- the host cell supports the replication and propagation of the vector.
- Host cells can be bacterial cells such as E. coli, or mammalian cells (e.g., cultured human cells or mouse cells).
- a suitable host cell such as a suitable mouse cell
- that cell line can be used to create a complete transgenic organism.
- Methods (i.e., means) for delivering vectors/constructs or other nucleic acids (such as in vitro transcribed RNA) into host cells such as bacterial cells and mammalian cells are well known to one of ordinary skill in the art, and are not provided in detail herein.
- any method for nucleic acid delivery into a host cell finds use with the compositions and methods described herein.
- methods for delivering vectors or other nucleic acid molecules into bacterial cells such as Escherichia coli are routine, and include electroporation methods and transformation of E. coli cells that have been rendered competent by previous treatment with divalent cations such as CaCh.
- Methods for delivering vectors or other nucleic acid (such as RNA) into mammalian cells in culture are routine, and a number of transfection methods find use with the compositions and methods described herein.
- lipid- based methods liposomes or lipoplexes
- Transfectamine® Life TechnologiesTM
- TransFectinTM Bio-Rad Laboratories
- cationic polymer transfections for example using DEAE- dextran
- direct nucleic acid injection biolistic particle injection
- viral transduction using engineered viral carriers (termed transduction, using e.g. , engineered herpes simplex virus, adenovirus, adeno-associated virus, vaccinia virus, Sindbis virus), and sonoporation.
- a “polypeptide” or “protein” is any polymer of amino acids (natural or unnatural, or a combination thereol), of any length, typically but not exclusively joined by covalent peptide bonds.
- a polypeptide can be from any source, e.g., a naturally occurring polypeptide, a polypeptide produced by recombinant molecular genetic techniques, a polypeptide from a cell, or a polypeptide produced enzymatically in a cell-free system.
- a polypeptide can also be produced using chemical (non-enzymatic) synthesis methods.
- a polypeptide is characterized by the amino acid sequence in the polymer.
- the term “peptide” typically refers to a small polypeptide, and typically is smaller than a protein. Unless otherwise stated, it is not intended that a polypeptide be limited by possessing or not possessing any particular biological activity.
- polypeptide is also intended to include the products of post-expression modifications of the naked amino acid sequence, i.e., modification of the nascent polypeptide, including without limitation and not limited to, glycosylation, acetylation, phosphorylation, amidation and derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non- naturally occurring amino acids.
- a polypeptide may be derived from a natural biological source or produced by recombinant technology, or produced by chemical synthesis.
- an "isolated" polypeptide or a fragment, variant, or derivative thereof is a polypeptide that is not in its natural milieu. No particular level of purification is required.
- an isolated polypeptide can be removed from its native or natural environment.
- Recombinant polypeptides and recombinant proteins expressed in host cells are considered isolated for purposes of the present disclosure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
- Polypeptides as described herein includes fragments, derivatives, analogs or variants of the foregoing polypeptides and any combinations thereof as well.
- fragment includes polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of the natural peptide, or sequences derived from a parent molecule that is also a recombinant polypeptide.
- the polypeptide fragments, derivatives, analogs or variants retain or improve upon the biological activity of the parent polypeptide.
- the term “recombinant” in reference to a nucleic acid or polypeptide indicates that the material (e.g., a recombinant nucleic acid, gene, polynucleotide, polypeptide, etc.) has been altered by human intervention, e.g., produced by recombinant DNA techniques.
- the arrangement of parts of a recombinant molecule is not a native configuration, or the primary sequence of the recombinant polynucleotide or polypeptide has in some way been manipulated.
- a naturally occurring nucleotide sequence becomes a recombinant polynucleotide if it is removed from the native location from which it originated (e.g., a chromosome), or if it is transcribed from a recombinant DNA construct.
- a gene open reading frame is a recombinant molecule if that nucleotide sequence has been removed from it natural context and cloned into any type of nucleic acid vector (even if that ORF has the same nucleotide sequence as the naturally occurring gene). Protocols and reagents to produce recombinant nucleic acids and polypeptides are well known to one of ordinary skill in the art.
- the term “recombinant cell line” refers to any cell line containing a recombinant nucleic acid, that is to say, a nucleic acid that is not native to that host cell.
- heterologous or “exogenous” as applied to polynucleotides or polypeptides refers to molecules that have been rearranged or artificially supplied to a biological system and are not in a native configuration (e.g., with respect to sequence, genomic position or arrangement of parts) or are not native to that particular biological system. These terms indicate that the relevant material originated from a source other than the naturally occurring source, or refers to molecules having a non-natural configuration, genetic location or arrangement of parts.
- exogenous and “heterologous” are sometimes used interchangeably with “recombinant.”
- the term “tag” as used in protein tags refers generally to peptide sequences that are genetically fused to other protein open reading frames, thereby producing recombinant fusion proteins. Ideally, the fused tag does not interfere with the native biological activity or function of the larger protein to which it is fused. Protein tags are used for a variety of purposes, for example but not limited to, tags to facilitate purification, detection or visualization of the fusion proteins. Depending on use, the terms “marker,” “reporter” and “tag” may overlap in definition, where the same protein or polypeptide can be used as either a marker, a reporter or a tag in different applications.
- a polypeptide may simultaneously function as a reporter and/or a tag and/or a marker, all in the same recombinant gene or protein.
- prokaryote refers to organisms belonging to the Kingdom
- Procarya generally distinguishable from eukaryotes by their unicellular organization, asexual reproduction by budding or fission, the lack of a membrane-bound nucleus or other membrane-bound organelles, a circular chromosome, the presence of operons, the absence of introns, message capping and poly-A mRNA, a distinguishing ribosomal structure and other biochemical characteristics.
- Prokaryotes include subkingdoms Eubacteria (“true bacteria”) and Archaea (sometimes termed “archaebacteria”).
- the terms “bacteria” or “bacterial” refer to prokaryotic Eubacteria, and are distinguishable from Archaea, based on a number of well- defined morphological and biochemical criteria.
- the term “eukaryote” refers to organisms (typically multicellular organisms) belonging to the Kingdom Eucarya, generally distinguishable from prokaryotes by the presence of a membrane-bound nucleus and other membrane-bound organelles, linear genetic material (i.e., linear chromosomes), the absence of operons, the presence of introns, message capping and poly- A mRNA, a distinguishing ribosomal structure and other biochemical characteristics.
- the terms “mammal” or “mammalian” refer to a group of eukaryotic organisms in the phylum Chordata that are endothermic amniotes distinguishable from other Chordata by a number of physical, physiological and molecular traits.
- the group mammals includes the orders Rodentia (including mice and rats) and primates (including humans).
- the term “encode” refers broadly to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first.
- the second molecule may have a chemical structure that is different from the chemical nature of the first molecule.
- the term “encode” describes the process of semi conservative DNA replication, where one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase.
- a DNA molecule can encode an RNA molecule (e.g., by the process of transcription that uses a DNA-dependent RNA polymerase enzyme).
- an RNA molecule can encode a polypeptide, as in the process of translation.
- the term “encode” also extends to the triplet codon that encodes an amino acid.
- an RNA molecule can encode a DNA molecule, e.g., by the process of reverse transcription incorporating an RNA-dependent DNA polymerase.
- a DNA molecule for example a gene, can encode a polypeptide, where it is understood that “encode” as used in that case incorporates both the processes of transcription and translation.
- the term “derived from” refers to a process whereby a first component
- a first molecule e.g., a first molecule
- information from that first component is used to isolate, derive or make a different second component (e.g., a second molecule that is different from the first).
- variant refers to a first composition (e.g., a first molecule), that is related to a second composition (e.g., a second molecule, also termed a “parent” molecule).
- the variant molecule can be derived from, isolated from, based on or homologous to the parent molecule.
- the term variant can be used to describe either polynucleotides or polypeptides.
- a “variant” polypeptide can refer to a sequence variant where the variant molecule has an amino acid sequence that is not identical to the patent molecule, for example, because the variant molecule contains targeted amino acid substitutions.
- a variant molecule can, for example, have 100% amino acid sequence identity with the original parent molecule and comprise additional amino acid residues, or alternatively, can have less than 100% amino acid sequence identity with the parent molecule.
- a variant of a parent amino acid sequence can be a second amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or any number less than 100% identical in amino acid sequence compared to the original parent amino acid sequence.
- Polypeptide variants also include polypeptides comprising the entire parent polynucleotide, and further comprising additional fused amino acid sequences.
- Polypeptide variants also include polypeptides that are portions or subsequences of the parent polypeptide, for example, unique subsequences (e.g., as determined by standard sequence comparison and alignment techniques).
- polypeptide variants include amino acid sequences that contain minor, trivial or inconsequential changes relative to the parent sequence.
- minor, trivial or inconsequential changes include changes to an amino acid sequence that (i) result in substitutions, deletions or insertions that have little or no impact on the biological activity of the polypeptide, and (ii) result in the substitution of an amino acid with a chemically similar amino acid, i.e., conservative amino acid substitutions.
- Polypeptide variants include polypeptides comprising the entire parent polypeptide, and further comprising additional fused amino acid sequences. Polypeptide variants also includes polypeptides that are portions or subsequences of the parent polypeptide, for example, unique subsequences (e.g., as determined by standard sequence comparison and alignment techniques) of the polypeptides disclosed herein are also encompassed by the compositions and methods described herein.
- polypeptide variants includes polypeptides that contain minor, trivial or inconsequential changes to the parent amino acid sequence.
- minor, trivial or inconsequential changes include amino acid changes (including substitutions, deletions and insertions) that have little or no impact on the biological activity of the polypeptide, and yield functionally identical polypeptides, including additions of non-functional peptide sequence.
- the variant polypeptides change the biological activity of the parent molecule.
- variant polypeptides can be derived from the polypeptides provided by the present disclosure.
- variant polynucleotides are also provided by the present disclosure.
- variant polynucleotides are any polynucleotide that encodes a variant polypeptide.
- a variant polynucleotides can have entire nucleotide sequence identity with the original parent polynucleotide molecule and contain additional sequence information, or alternatively, can have less than 100% nucleotide sequence identity with the parent molecule.
- a variant of a parent nucleotide sequence can have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or any value less than 100% identity with the patent nucleotide sequence.
- Polynucleotide variants also include polynucleotides comprising the entire parent polynucleotide, and further comprising additional fused nucleotide sequences.
- Polynucleotide variants also includes polynucleotides that are portions or subsequences of the parent polynucleotide, for example, unique subsequences (e.g., as determined by standard sequence comparison and alignment techniques). [0148] In another aspect, polynucleotide variants includes nucleotide sequences that contain minor, trivial or inconsequential changes to the parent nucleotide sequence.
- nucleotide sequence that (i) do not change the amino acid sequence of the corresponding polypeptide, e.g., substitutions in codon wobble positions, (ii) occur outside the protein-coding open reading frame of a polynucleotide, (iii) result in deletions or insertions that may impact the corresponding amino acid sequence, but have little or no impact on the biological activity of the polypeptide, (iv) the nucleotide changes result in the substitution of an amino acid with a chemically similar amino acid, i.e., conservative amino acid substitutions.
- variants of that polynucleotide can include nucleotide changes that do not result in loss of function of the polynucleotide.
- conservative variants of the disclosed nucleotide sequences that yield functionally identical nucleotide sequences are encompassed by the compositions and methods described herein.
- One of skill will appreciate that many variant polynucleotides can be derived from the disclosed nucleotide sequences.
- polynucleotide or polypeptide variants can include variant molecules that alter, add or delete a small percentage of the nucleotide or amino acid positions, for example, typically less than about 10%, less than about 5%, less than 4%, less than 2% or less than 1%.
- nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same (“identical”) or have a specified percentage of amino acid residues or nucleotides that are identical (“percent identity”) when compared and aligned for maximum correspondence with a second molecule, as measured using a sequence comparison algorithm (e.g., by a BLAST alignment, or any other algorithm known to persons of skill), or alternatively, by visual inspection.
- sequence comparison algorithm e.g., by a BLAST alignment, or any other algorithm known to persons of skill
- nucleic acids or polypeptides refers to two or more sequences or subsequences that have at least about 60%, about 80%, about 90%, about 90-95%, about 95%, about 98%, about 99% or more nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence using a sequence comparison algorithm or by visual inspection.
- sequence comparison algorithm or by visual inspection.
- substantially identical sequences are typically considered to be “homologous,” without reference to actual ancestry.
- the “substantial identity” between nucleotides exists over a region of the polynucleotide at least about 50 nucleotides in length, at least about 100 nucleotides in length, at least about 200 nucleotides in length, at least about 300 nucleotides in length, or at least about 500 nucleotides in length, most preferably over their entire length of the polynucleotide.
- the “substantial identity” between polypeptides exists over a region of the polypeptide at least about 50 amino acid residues in length, more preferably over a region of at least about 100 amino acid residues, and most preferably, the sequences are substantially identical over their entire length.
- sequence similarity in the context of two polypeptides refers to the extent of relatedness between two or more sequences or subsequences. Such sequences will typically have some degree of amino acid sequence identity, and in addition, where there exists amino acid non identity, there is some percentage of substitutions within groups of functionally related amino acids. For example, substitution of a serine with a threonine in a polypeptide is sequence similarity, but not identity.
- homologous refers to two or more polypeptides when they are derived, naturally or artificially, from a common ancestral polypeptide.
- nucleotide sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid. Homology in proteins is generally inferred from amino acid sequence identity and sequence similarity between two or more proteins. The precise percentage of identity and/or similarity between sequences that is useful in establishing homology varies with the polynucleotide and polypeptide at issue, but as little as 25% sequence similarity is routinely used to establish homology.
- sequence similarity percentages e.g., BLASTP and BLASTN using default parameters
- portion refers to any portion of a larger sequence (e.g., a nucleotide subsequence or an amino acid subsequence) that is smaller than the complete sequence from which it was derived.
- the minimum length of a subsequence is generally not limited, except that a minimum length may be useful in view of its intended function.
- the subsequence can be derived from any portion of the parent molecule. In some aspects, the portion or subsequence retains a critical feature or biological activity of the larger molecule, or corresponds to a particular functional domain of the parent molecule, for example, the DNA-binding domain, or the transcriptional activation domain.
- an assay for direct amplification and qualitative detection of target RNA molecules and sequences, e.g., from various eukaryotic, prokaryotic, and viral pathogens, in test samples, such as patient body fluids or tissue samples.
- the assay is a real time assay.
- the assay is a multiplex assay that can detect multiple pathogens.
- the assay is a multiple, real time assay.
- the assay disclosed herein can be also employed for detection of a mutation in the genomic RNA.
- the assay can be employed for detection of one or more mutations e.g., single-nucleotide polymorphisms (SNPs), in a genomic RNA.
- the multiplex real time assay within the disclosure relates to an RNA replicase-dependent amplification (RDA) of target RNA molecules using an RNA replicase, also known as an RNA-dependent RNA polymerase or RdRP, and detection of the amplified target RNA molecules and sequences for molecular diagnosis of various pathogens in body fluid or tissue samples or a mutation in the genomic RNA.
- RDA RNA replicase-dependent amplification
- the in vitro diagnostic assay is directed towards the diagnosis of various eukaryotic, prokaryotic, and viral pathogens, in particular, viral, bacterial and/or parasitic infections, in subjects, particularly human subjects and patients, or a mutation in the genomic RNA.
- the assay may also provide differential detection of the presence or absence of multiple pathogens in a single assay.
- the assay may also provide differential detection of the presence or absence of a pathogen by detecting multiple targets to the pathogen in a single assay.
- the direct RNA amplification assays disclosed herein can be performed in less than 3 hours, in some cases less than 2.5 hours, and in some cases even in less than 75 minutes.
- the reagents employed in the assays are provided in solid form, for example in a lyophilized form. In one embodiment, the reagents employed in the assays are provided in solution form, for example in a frozen liquid form. In some embodiments, the different reagents employed in the assays are provided in various different forms, for example, some reagents are provided in solid forms, for example in a lyophilized form, whereas some other reagents are provided in solution forms, for example in a frozen liquid forms. In some embodiments, the reagents employed in the assays are provided in a single container.
- the reagents employed in the assays are provided in separate containers, for example, in two, three, four, five or more containers.
- the reagents can be simply combined with the liquid sample.
- reagents provided in solid form can be simply rehydrated using a rehydration solution, for example, a rehydration solution provided as a reagent in one of the containers, and combined with the liquid sample.
- the assay can thus be carried out with a minimal amount of transfer of reagent solutions, greatly reducing the possibility of contamination or loss of sample, as well as the time needed for completion of the assay.
- the reagents and components of the amplification mixtures, amplification compositions, compositions and the like employed herein for direct amplification and detection of a target can include:
- RNA replicase RNA-dependent RNA polymerase
- NTPs nucleoside triphosphates
- RNAse inhibitor optionally, an RNAse inhibitor, one or more of a reaction helper protein, or combinations thereof.
- the target RNA molecules and sequences that can be amplified and detected by employing the multiplex real time PCR-based assays within the disclosure, in particular, by the RNA replicas e-dependent amplification (RDA) disclosed herein include, but are not limited to, viral RNAs, mRNAs, and other non-coding RNAs, such as rRNAs, tRNAs, snRNAs, etc.
- a sample containing a target RNA molecule or sequence can be prepared, as described further below, combined with or added to the amplification mixtures, amplification compositions, and compositions described herein, and RNA amplicons of the target RNA molecule or sequence can be produced using, for example, an RNA replicase-dependent amplification (RDA) of the target RNA molecule or sequence, if and when such target RNA is present in the sample.
- RDA RNA replicase-dependent amplification
- An amplicon can refer to a piece of a genetic fragment or nucleic acid sequence, e.g., an RNA molecule or sequence, which can be the source and/or product of amplification or replication events.
- an RNA amplicon refers to the product of a RNA amplification reaction, in particular, an RNA replicase-dependent amplification (RDA) of the target RNA molecule or sequence, which within various embodiments of the disclosure can be performed under isothermal conditions.
- the RNA amplicons that can be produced by the RDA disclosed herein include single-stranded RNA amplicons (ssRNA amplicons), double-stranded RNA amplicons (dsRNA amplicons), or combinations thereof.
- the compositions described herein include an enzyme that possesses an RNA polymerase activity for amplification of an RNA molecule or sequence (e.g.
- the enzyme can be an RNA replicase, also known as an RNA-dependent RNA polymerase or RdRP, which possesses an RNA polymerase activity and is capable of direct amplification of RNA molecule or sequence (e.g. a target RNA molecule or sequence from a pathogen) without formation of a cDNA or DNA.
- the RNA replicase enzyme is further capable of performing the amplification of RNA, also referred to as the RNA replicase-dependent amplification (RDA) of RNA, essentially isothermally.
- the RNA replicase is derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV), and comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
- the RNA replicase is a recombinant RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV).
- the recombinant RNA replicase comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
- the composition can include one or more stabilizers.
- nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 is disclosed herein.
- a nucleic acid encoding the amino acid sequence of SEQ ID NO: 3, is also disclosed herein.
- Exemplary nucleic acid sequences of the RNA replicase of the various embodiments disclosed herein can include the nucleic acid sequence set forth in SEQ ID NO: 2, or a nucleic acid sequence having at least 80%, 90%, or 95% sequence identity thereto, and SEQ ID NO: 4, or a nucleic acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
- NTPs nucleoside triphosphates
- RNA contains four different nitrogenous bases: adenine (A), guanine (G), and cytosine (C), and uracil (U).
- RNA synthesis is catalyzed by RNA polymerases that utilizes the NTPs as substrates to synthesize the RNA chain.
- the four different NTPs ATP, CTP, GTP and UTP are each present in equal concentrations that is appropriate for the assays disclosed herein.
- the four different NTPs ATP, CTP, GTP and UTP are each present in different concentrations that is appropriate for the assays disclosed herein.
- such reagents may include an RNAse inhibitor (New England
- compositions described herein can also include one or more reaction helper proteins.
- the reaction helper protein can be a protein that assists in the activity of the RNA replicase (RNA-dependent RNA polymerase), stabilizes the target RNA, or separates a double- stranded RNA into single-stranded RNA.
- the reaction helper protein can be an RNA-binding protein (RBP), which includes, but is not limited to, an RNA chaperone, an RNA helicase, an RNA binding-factor, a scaffold protein, or an RNA-modifying enzyme that may be required for, but not limited to, an RNA replicase activity, target RNA-binding, and/or assisting in stability of a target RNA.
- RBP RNA-binding protein
- the RNA helicase can be used to separate strands in a duplex (i.e., double-stranded RNA), allowing primers to bind the duplex and be extended during the amplification process.
- compositions described herein can further include any reagent that is conventionally employed for nucleic acid amplification, more specifically, for direct and/or isothermal RNA amplification utilizing RDA, including those that are not explicitly disclosed herein.
- exemplary primers are designed, and amplification conditions selected, such that efficient RNA amplification, e.g., RNA replicase-dependent amplification (RDA), and detection of one or more target RNA molecules and sequences can be performed simultaneously using the same amplification conditions.
- efficient RNA amplification e.g., RNA replicase-dependent amplification (RDA)
- RDA RNA replicase-dependent amplification
- Exemplary primer sets having this property are described below.
- compositions described herein include at least one target oligonucleotide primer specific for a target RNA molecule or sequence to be detected (analyte), e.g., an RNA molecule or sequence derived from a pathogen.
- target oligonucleotide primer or primers are specific for selected primer binding sites, e.g., in the RNA sequence, which can be in highly conserved regions of a genome, e.g., genome of the pathogen, to be detected.
- the composition can include a single target oligonucleotide primer that is capable of performing successful asymmetric amplification of RNA.
- a single target oligonucleotide primer may be employed for avoiding generation of double-stranded RNA amplicons (dsRNA amplicons), while specifically generating single-stranded RNA amplicons (ssRNA amplicons) that can be advantageous for employing certain RNA amplicon detection methodologies, e.g., real-time and end-point RNA amplicon detection methodologies.
- the composition can include an oligonucleotide primer pair comprising a first target oligonucleotide primer and a second target oligonucleotide primer.
- the first and second target oligonucleotide primers are provided in the composition in equal concentrations.
- the first and second target oligonucleotide primers are provided in the composition in unequal concentrations.
- the first target oligonucleotide primer can be present at a concentration that is at least two times, at least three times, at least four time, or at least five times greater than the concentration of the second target oligonucleotide primer.
- the second target oligonucleotide primer is provided at concentration within a range of about 25 nM to about 5 mM, including at least about 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 75 nM, 100 nM, 150 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 mM, 1.5 mM , 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, etc.
- compositions described herein include an oligonucleotide primer pair comprising a first target oligonucleotide primer and a second target oligonucleotide primer that are specific for a double-stranded target RNA to be detected.
- the first and second target oligonucleotide primers can hybridize to opposite strands of the double-stranded RNA (dsRNA) in 5’ to 3’ orientation for the amplification process.
- the composition can further include a control oligonucleotide primer or primer pair specific to a process control sequence, e.g., a process control RNA sequence, and optionally, a control probe for detection of the process control sequence.
- a control oligonucleotide primer or primer pair specific to a process control sequence e.g., a process control RNA sequence
- a control probe for detection of the process control sequence e.g., a process control RNA sequence
- the target oligonucleotide primer or primer pair and control oligonucleotide primer or primer pairs are designed such that amplification and detection of the target RNA and the process control sequence, can be performed simultaneously using the same amplification conditions.
- the primers are designed such that the annealing/melting temperatures of primer/binding site duplexes for one or more target RNA sequences (e.g., analytes) and for the process control sequence, are approximately equivalent, e.g., within 3 °C, within 2 °C, or within 1 °C or less, in the amplification reaction environment.
- the annealing/melting temperatures of primer/binding site duplexes for one or more target RNA sequences (e.g., analytes) and for the process control sequence are within 5 °C, or are nearly equivalent, e.g., within 0.5 °C or less.
- a target RNA sequence of a pathogen with high sequence variability e.g., of a viral pathogen with high sequence variation
- multiple variations of a primer sequence may be required to adequately detect all possible variations of the viral target RNA sequences.
- R A/G
- Y C/G
- W A/T
- S C/G
- K G/T
- a few different versions of a primer sequence may be used for effective detection of all possible variations of a pathogenic target RNA sequences.
- one primer may be designed to be longer, shorter, or shifted as compared to the other primers for effective detection of all possible variations of a pathogenic target RNA sequences.
- the oligonucleotide primer or oligonucleotide primer pairs of the disclosure are preferably specific to or specific for selected primer binding sites in the genomic target RNA sequence (e.g., for detecting mutations in the genomic RNA) or to an RNA sequence of the target pathogen (e.g., for detecting presence of target pathogen(s) in a sample).
- the oligonucleotide primer or oligonucleotide primer pairs of the disclosure are preferably specific to regions in the genome of the target pathogen(s), in particular, to highly conserved regions in the genome of the target pathogen(s) described in detail below.
- the primers are substantially complementary, and in some embodiments exactly complementary, to selected regions of regions, including highly conserved regions, of RNA, such as viral, bacterial or parasitic RNA.
- the sequences of the highly conserved regions used for primer design may be consensus sequences derived from multiple strains and/or subtypes of a pathogen.
- the primers and probes of various embodiments herein may be subject to various modification, such as fluorescent or chemiluminescent-labeling, and biotinylation.
- Amplified nucleic acid products e.g., RNA amplicons, may be detected by various known methods including end-point ethidium-bromide staining, or by detecting the amplified products by means of a label selected from a radiolabel, a fluorescent-label, and an enzyme.
- Labeling methods may also include radioactive isotopes, chromophores, and ligands such as biotin or haptens, which can be readily detected by reaction with labeled forms of their specific binding partners, e.g., avidin and antibodies respectively.
- the probes are RNA probes that include, but are not limited to, FITC, DIG, or DNP labeled probes, which bind to the RNA amplicons, for example, to single-stranded amplicons or to one or both strands of double-stranded amplicons.
- compositions described herein can also include specific
- RNA probes labeled with a quencher on one end and a fluorophore reporter on the other end for binding to the RNA amplicons and in some embodiments, to biotinylated RNA amplicons.
- the quencher is separated from the fluorophore reporter on the probe, thereby de-quenching the fluorophore and resulting in an increase in the detectable fluorescent signal.
- specific RNA probes labeled with a quencher on one end and a fluorophore reporter on the other end for binding to the RNA amplicons may include a hairpin loop between the fluorophore and the quencher.
- RNA probes labeled with a quencher on one end and a fluorophore reporter on the other end for binding to the RNA amplicons may not include a hairpin loop between the fluorophore and the quencher.
- the probe without a hairpin loop may need to be hydrolyzed by a polymerase (e.g., TaqMan) or enzymatically cut in order to un-quench the fluorophore from the quencher.
- a polymerase e.g., TaqMan
- additional fluorophore reporter molecules are separated from their quenchers, resulting in additional signal.
- the sample can be reported as positive for the detected RNA sequence. Accordingly, in some embodiments, the RNA amplification and detection assays within the disclosure can provide for real-time detection of a target RNA sequence using such fluorescent- labeled probes.
- the compositions described herein can include primers, e.g., biotinylated primers, which bind to target RNA sequences that have been separated during the amplification process.
- the compositions described herein can further include labeled RNA probes, e.g., FITC, DIG, or DNP labeled probes, which bind to the biotinylated RNA amplicons, for example, to the single-stranded biotinylated amplicons or to one or both strands of double-stranded biotinylated amplicons.
- the biotinylated amplicons can bind to streptavidin coated beads and allow for end-point detection of the amplicons on surfaces coated with anti-FITC antibodies.
- the compositions described herein can include a detectable probe for detection of the target RNA, including detection of the RDA amplified RNA amplicons.
- the detectable probes can hybridize to the RNA target sequence and/or to one or more of the RNA amplicons that are produced during the RNA amplification, e.g., RDA, of the target RNA, if the target RNA is present in the sample, and thereby allows for detection of presence or absence of the target RNA in the sample.
- the compositions described herein can also include a control probe for detection of the process control.
- the detectable probe can be an optically-labeled probe, for example, an optically-labeled probe that includes a fluorescent compound.
- the RNA amplification is a real-time amplification, and the RNA amplicons produced during the amplification is detected real-time.
- the RNA amplicons produced during the RNA amplification can be determined by an end-point analysis, for example, an end-point analysis achieved by a lateral-flow using, for example, biotinylated primers, RNA probes, e.g., FITC, DIG, or DNP labeled probes (that bind to the RNA amplicons and biotinylated RNA amplicons), and anti-FITC antibodies, as described above.
- end-point analysis for example, an end-point analysis achieved by a lateral-flow using, for example, biotinylated primers, RNA probes, e.g., FITC, DIG, or DNP labeled probes (that bind to the RNA amplicons and biotinylated RNA amplicons), and anti-FITC antibodies, as described above.
- RDA RNA Reylicase-Deyendent Amplification
- Unequal concentrations of oligonucleotide primers can be used to preferentially generate single-stranded amplicon for hybridization to a probe for detection of a target nucleic acid segment, e.g. an RNA.
- asymmetric RDA may be performed isothermally at a temperature in the range of about 20 °C and 75 °C, within a range of 35 °C and 70 °C, or more specifically, within a range of 60 °C and 66°C for a thermophilic RDA reaction.
- the first or forward oligonucleotide primer can be provided at a higher concentration, e.g., at least three fold excess, than the second or reverse oligonucleotide primer, which serves as the limiting primer.
- the second or reverse oligonucleotide primer can be provided in at least three-fold excess where the first or forward oligonucleotide primer serves as the limiting primer. Depletion of the limiting primer during exponential amplification results in linear synthesis of the product generated from the excess primer. This leads to accumulation of the single-stranded amplicon that originated from the excess primer.
- the limiting primer can be provided in the range of about 25 nM to about 5 mM, or in the range of about 25 nM to about 75 nM (e.g., about 50 nM). In various embodiments, the excess primer can be provided at a concentration that is in the range of approximately 1.5 to 10 times that of the limiting primer (for example, 200 nM). In one embodiment, the excess primer can be provided at a concentration that is three or four times higher than the concentration of the limiting primer.
- the primers are designed, and amplification conditions selected, such that efficient amplification, e.g., RNA replicase-dependent amplification (RDA), and detection of one or more target RNA molecules and sequences can be performed simultaneously using the same thermal cycling conditions on a thermocycler.
- efficient amplification e.g., RNA replicase-dependent amplification (RDA)
- detection of one or more target RNA molecules and sequences can be performed simultaneously using the same thermal cycling conditions on a thermocycler.
- the primers are designed, and amplification conditions are selected, such that efficient amplification, e.g., RNA replicase-dependent amplification (RDA), and detection of one or more target RNA molecules and sequences can be performed simultaneously using the same isothermal conditions, for example in the range of 20 °C and 75 °C, within a range of 35 °C and 70 °C, or within a range of 60 °C and 66°C, and essentially without a thermocycler and under isothermal conditions.
- efficient amplification e.g., RNA replicase-dependent amplification (RDA)
- RDA RNA replicase-dependent amplification
- a multiplex RNA amplification reaction can be carried out under optimized conditions in a single vessel, or in multiple vessels but under the same RNA amplification conditions, generating amplicons for one or more of the target pathogens present in a sample.
- the primers used in assays disclosed herein are designed such that the annealing/melting temperatures of primer/binding site duplexes for one or more target RNA sequences (e.g., analytes) and for the process control sequence, are approximately equivalent, e.g., within 3 °C, within 2 °C, or within 1 °C or less, in the amplification reaction environment.
- the annealing/melting temperatures of primer/binding site duplexes for one or more target RNA sequences (e.g., analytes) and for the process control sequence are within 5 °C, or are nearly equivalent, e.g., within 0.5 °C or less.
- Applications of this method can include detection and diagnosis of a pathogen in a sample (e.g., a clinical or test sample), detecting a mutation in a genomic RNA (e.g., human genomic RNA), and single polynucleotide detection.
- a pathogen e.g., a pathogen in a sample
- detecting a mutation in a genomic RNA e.g., human genomic RNA
- single polynucleotide detection e.g., single polynucleotide detection.
- the current multiplex and/or real time assays within the disclosure are useful for detection and diagnosis of various pathogens, for example, various eukaryotic, prokaryotic, and viral pathogens, in clinical samples, such as body fluids or tissue samples.
- the in vitro diagnostic assays within the disclosure relates to direct amplification of target RNA molecules (i.e., analytes) using an RNA replicase-dependent amplification (RDA) and detection of the amplified target RNA molecules for molecular detection of a mutation in a genomic RNA, as well as diagnosis of various pathogens, in particular, viral, bacterial and/or parasitic infections, in test samples from subjects, particularly human subjects and patients.
- RDA RNA replicase-dependent amplification
- pathogens in particular, viral, bacterial and/or parasitic infections
- Viral pathogens of significance may include, for example, viruses that use cognate
- viral pathogens may include, for example, a positive-strand genomic RNA virus, a negative-strand genomic RNA virus, and a double-strand genomic RNA virus.
- viral pathogens may include, for example, the human immunodeficiency virus (HIV), the hepatitis C virus (HCV), coronavirus, Ebola virus, the hepatitis A virus, the flu viruses (influenza A, B and C), Avian influenza virus, the SARS virus, the polio virus (Poliovirus), the measles virus, the mumps virus, the rubella virus, rotavirus, sapovirus, norovirus, human respiratory syncytial virus (RSV) A and B, human metapneumo virus (hMPV), herpes simplex virus 1 and 2 (HSV-1 and HSV-2), human parainfluenza virus (HPIVs) 1-4, Varicella-zoster virus (VZV, also referred to as HSV-3.
- HSV human immunodeficiency virus
- HCV hepatitis C virus
- coronavirus Ebola virus
- Ebola virus the hepatitis A virus
- the flu viruses
- Pathogens of significance may also include, but are not limited to, Clostridium difficile
- C. diff various Staphylococcus species, such as methicillin-resistant Staphylococcus aureus (MRSA), healthcare-associated MRSA (HA-MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant coagulase-negative staphylococci (MRCNS), methicillin-sensitive coagulase-negative staphylococci (MSCNS), methicillin-resistant Staphylococcus epidermidis (MRSE) and methicillin-sensitive Staphylococcus epidermidis (MRSE), Group B streptococcus, Bordetella pertussis, Bordetella parapertussis, Bordetella holmesii, and parasites such as, Cryptosporidium species, Entamoeba species including E.
- MRSA methicillin-resistant Staphylococcus aureus
- H-MRSA healthcare-associated MRSA
- MSSA methicillin-sensitive Staphylococcus aureus
- pathogens may include a mycobacterium species including, but not limited to, Mycobacterium kansasii, Mycobacterium tuberculosis, Mycobacterium intracellualre, Mycobacterium tuberculosis , Mycobacterium avium, and Mycoplasma pneumonia.
- pathogens may include parasitic helminth and protozoan parasites including, but not limited to, Acanthamoeba species, Anisakis species, Ascaris lumbricoides , Botfly, Balantidium coli, Bedbugs, Cestoidea (tapeworms), Chiggers, Cochliomyia hominivorax, Cryptosporidium species, Entamoeba species including E. histolytica, Fasciola hepatica and other liver flukes, Giardia species (e.g., G.
- parasitic helminth and protozoan parasites including, but not limited to, Acanthamoeba species, Anisakis species, Ascaris lumbricoides , Botfly, Balantidium coli, Bedbugs, Cestoidea (tapeworms), Chiggers, Cochliomyia hominivorax, Cryptosporidium species, Entamoeba species including E. histolytica, Fas
- the test sample may be any clinical sample, e.g., any body fluid or tissue sample including, but not limited to, mammalian blood, serum, plasma, or cells, suspected of containing a target pathogen, collected according to procedures known in the art.
- a target pathogen e.g., respiratory viruses
- respiratory viruses may be detected in a nasal swab, nasophyrangeal swab, or nasal aspirate/wash specimens.
- the target pathogen(s) may be detected from a stool sample.
- Extraction of nucleic acids, e.g., RNA, from the test sample may be performed manually or automatically, as known in the art, using the appropriate reagents and following the manufacturer's instructions for automated systems.
- Automated sample extraction platforms include, for example, the NucliSENS® easyMag® system (bioMerieux). In some embodiments, no extraction step is needed or performed.
- a process control can be added to an aliquot of every specimen prior to the extraction procedure.
- the process control serves to assure adequate nucleic acid extraction and to reflect the presence of any inhibitors that may be present in the sample.
- a solid reagent composition used such reagent is rehydrated using a rehydration solution, and aliquots are placed in amplification reaction tubes or plate wells. Aliquots of prepared fluid sample, containing nucleic acids and process control, are then added. Alternatively, the rehydrated reagents can be added to the fluid sample. Amplification, e.g., RDA, is then carried out in a thermal cycling apparatus, or without requiring thermocy cling under isothermal conditions.
- the rehydration solution may be composed of a multivalent cation cofactor for catalyzing enzymes and reagents to maintain an optimal pH buffer for an efficient amplification reaction.
- the rehydration solution comprises an enzyme catalyzing cofactor, such as Mg +2 , Mn +2 , or other multivalent cations, which is provided as a cation compound (such as magnesium chloride (MgCh), manganese acetate (Mh(OA ), or any other cation compound) in the rehydration solution at a concentration that is suitable for efficient amplification.
- an enzyme catalyzing cofactor such as Mg +2 , Mn +2 , or other multivalent cations
- MgCh magnesium chloride
- Mh(OA OA manganese acetate
- any other cation compound such as magnesium chloride (MgCh), manganese acetate (Mh(OA ), or any other cation compound
- a method for detecting a target RNA in a sample comprising:
- RNA replicase RNA-dependent RNA polymerase
- NTPs nucleoside triphosphates
- RNAse inhibitor optionally, an RNAse inhibitor, one or more of a reaction helper protein, or combinations thereof;
- RNA replicase-dependent amplification RDA
- the methods disclosed herein relates to direct and/or isothermal amplification of RNA.
- the methods comprises direct amplification of RNA, i.e., RNA replicase-dependent amplification (RDA), without formation of a cDNA or DNA.
- RDA RNA replicase-dependent amplification
- the methods comprises direct amplification of RNA that is performed essentially isothermally.
- the method utilizes an RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) that comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
- NS5B nonstructural protein 5B
- HCV hepatitis C virus
- the method utilizes a recombinant RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV).
- the recombinant RNA replicase comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
- a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 is disclosed herein.
- a nucleic acid encoding the amino acid sequence of SEQ ID NO: 3, is also disclosed herein.
- Exemplary nucleic acid sequences of the RNA replicase of the various embodiments disclosed herein can include the nucleic acid sequence set forth in SEQ ID NO: 2, or a nucleic acid sequence having at least 80%, 90%, or 95% sequence identity thereto, and SEQ ID NO: 4, or a nucleic acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
- compositions employed in the methods disclosed herein can further include any reagent that is conventionally employed for nucleic acid amplification, more specifically, for direct and/or isothermal RNA amplification utilizing RDA, including those that are not explicitly disclosed herein.
- kits comprising the compositions described above.
- the kit comprises an RNA replicase (RNA-dependent RNA polymerase), at least one target oligonucleotide primer specific for a target RNA to be amplified, nucleoside triphosphates (NTPs) (i.e., ATP, CTP, GTP and UTP), one or more detectable, target probe(s) for detection of the target RNA, and optionally, an RNAse inhibitor, one or more reaction helper protein, or combinations thereof.
- NTPs nucleoside triphosphates
- the reaction helper protein is a protein that assists in the activity of the RNA replicase (RNA-dependent RNA polymerase), stabilizes the target RNA, or separates a double-stranded RNA into single-stranded RNA.
- the reaction helper protein can be an RNA-binding protein (RBP), which includes, but is not limited to, an RNA chaperone, an RNA helicase, an RNA binding-factor, a scaffold protein, or an RNA-modifying enzyme that may be required for, but not limited to, an RNA replicase activity, target RNA-binding, and/or assisting in stability of a target RNA.
- the RNA helicase can be used to separate strands in a duplex (i.e., double-stranded RNA), allowing primers to bind the duplex and be extended during the amplification process.
- compositions of the kit when combined with a sample
- compositions of the kit allow for a direct amplification i.e., RNA replicase-dependent amplification (RDA), of a target RNA without formation of a cDNA or DNA.
- RDA RNA replicase-dependent amplification
- the compositions of the kit allow for direct amplification of a target RNA that is performed essentially isothermally.
- the compositions of the kit includes an RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) that comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
- the compositions of the kit includes a recombinant RNA replicase that is derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV).
- the compositions of the kit includes a recombinant RNA replicase that comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
- nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 is disclosed herein.
- a nucleic acid encoding the amino acid sequence of SEQ ID NO: 3, is also disclosed herein.
- Exemplary nucleic acid sequences of the RNA replicase of the various embodiments disclosed herein can include the nucleic acid sequence set forth in SEQ ID NO: 2, or a nucleic acid sequence having at least 80%, 90%, or 95% sequence identity thereto, and SEQ ID NO: 4, or a nucleic acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
- the kit comprises a first container containing the aforesaid composition, and a second container containing a master mix. In some embodiments, the kit comprises a third container containing a process buffer. In some embodiments, the kit comprises a fourth container containing reagents related to process control. In some embodiments, the compositions of the kit are provided in lyophilized form, frozen liquid form, or combinations thereof. In some embodiments, the kit comprises a fifth container containing a rehydration solution. The kit may also contain instructions for using the aforesaid components and compositions in carrying out the amplification and detection assays disclosed herein.
- kits in a single container, containing components of an exemplary reagent composition as described above, e.g., an RNA replicase (RNA-dependent RNA polymerase), at least one target oligonucleotide primer specific for a target RNA to be amplified, nucleoside triphosphates (NTPs) (i.e., ATP, CTP, GTP and UTP), a detectable, target probe for detection of the target RNA, optionally, an RNAse inhibitor, a reaction helper protein, or combinations thereof, and, in some embodiments, one or more stabilizers.
- NTPs nucleoside triphosphates
- the components of an exemplary reagent composition can be provided as a solid composition in a first container in the kit.
- a container within the kit contains a rehydration solution, for use in rehydrating the solid composition.
- the rehydration solution may be composed of a multivalent cation cofactor for catalyzing enzymes.
- the rehydration solution comprises an enzyme catalyzing cofactor, such as Mg +2 , Mn +2 , or other multivalent cations, which is provided as a cation compound (such as magnesium chloride (MgCh). manganese acetate (Mh(OA ), or any other cation compound) in the rehydration solution at a concentration that is suitable for efficient amplification.
- the cation compound is provided at a concentration in the rehydration solution of between about 0.1-20 mM, 0.1-10 mM, 0.5- 10 mM, 0.5-8 mM, 0.5-6 mM, 0.1-6 mM, 0.1-5 mM, 0.5-5 mM, 0.1-3 mM, or 0.5-3 mM.
- the concentration of manganese acetate in the final assay is between about 0.1-20 mM, 0.1-10 mM, 0.5-10 mM, 0.5-8 mM, 0.5-6 mM, 0.1-6 mM, 0.1-5 mM, 0.5-5 mM, 0.1-3 mM, or 0.5-3 mM.
- the rehydration solution may also include reagents to maintain an optimal pH buffer for an efficient amplification reaction or optimal environment for any other proteins and enzymes involved in the methods described herein.
- the kit may contain a container containing a solution of the process control. External process controls for the pathogens being assayed may also be included.
- the kit may comprise a sample processing kit and a master mix container.
- the sample processing kit may comprise one or more containers comprising process buffer(s) for processing the sample.
- the master mix kit preferably comprises a container comprising a solid reagent composition, and another container comprising a rehydration solution, for use in rehydrating the solid reagent composition.
- Other components as described above may also be included such as, but not limited to, a container containing a process control solution and instructions.
- the solid reagent composition may correspond to any of the selected embodiments described above.
- the kit may also contain additional containers containing one or more process buffers.
- the kit may include a container containing a first process buffer and another container containing a second process buffer.
- the first process buffer comprises a sodium azide solution, NaOH, and lithium dodecyl sulfate.
- the second process buffer comprises a sodium azide solution, NaCl, Tris, EDTA, a control sequence, and water qs.
- the sample processing kit comprises a first and a second processing buffer, a solid reagent composition, and a rehydrating solution, each provided in a separate container.
- the kit includes software-driven assay protocols for use in commercial nucleic acid amplification instrumentations, when one is employed, which protocols may be provided on a CD.
- the kit may also contain instructions for using the aforesaid components and compositions in carrying out the amplification and detection assays disclosed herein.
- the present disclosure provides a wide variety of engineered and/or recombinant RNA replicase (RNA-dependent RNA polymerase) polypeptides, in particular, an RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV), where the engineered or recombinant molecules optionally comprise a wide array of tags, labels, markers and other types of fusion, modification moieties, and/or modifications such as deletions and additions of domains and segments.
- RNA replicase RNA-dependent RNA polymerase
- NSV nonstructural protein 5B
- HCV hepatitis C virus
- RNA replicase be limited by the use of any particular tags, labels, or fusion moieties recited herein, as one of skill recognizes that the use of these types of moieties and/or modifications is routine and well within the skill of the ordinary artisan, and further, one of skill will recognize and utilize still other reagents not specifically recited herein, and sill within the spirit of the disclosure.
- RNA replicases described herein are fusion polypeptides.
- the fusion polypeptides i.e., of the RNA replicases described herein, can comprise a signal sequence, a detectable moiety, an enzymatic detectable moiety, a detectable label (for example), a detectable particle, a fluorescent label, or any kind of polypeptide tag (for example, a purification tag, such as a poly-HIS tag).
- the RNA replicases described herein comprise a poly-histidine tag, in particular, a hexa-histidine peptide (HIS) tag, which provides for convenient purification of the fusion protein.
- polypeptide tags useful for purification include, but are not limited to, the hemagglutanin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein, GST, c-myc and the "flag" tag.
- HA hemagglutanin
- GST hemagglutanin
- c-myc a second amino acid sequence as a marker sequence or polypeptide tag to construct the fusion polypeptide.
- the appending of a second amino acid sequence as a marker sequence or polypeptide tag to construct the fusion polypeptide does not interfere with the specific activity, e.g., RNA amplification activity, of the modified RNA replicase.
- Polynucleotides and amino acid sequences encoding these fusion proteins are also encompassed by the disclosure.
- the disclosure provides RNA replicases having appended sequences to facilitate protein purification.
- the RNA replicases can be appended with coding sequence for a hexa-histidine peptide (HIS) tag, which allows for convenient purification of the fusion polypeptide, for example, using a Ni-NTA (nitrilotriacetic acid) resin, as known in the art.
- HIS hexa-histidine peptide
- Ni-NTA nitrilotriacetic acid
- the RNA replicase polypeptides can further comprise operably fused amino acid sequences that encode any number and any type of beneficial functionalities, for example, any type of fused polypeptide marker or tag of any desired sequence, or fused peptide sequences that mark the fusion protein for a particular post-translational modification.
- the disclosure provides numerous examples of protein markers and protein tags, any of which find use with the compositions and methods described herein.
- the compositions and methods described herein are not to be limited to polypeptide markers or tags recited herein, as one of ordinary skill recognizes the diversity of markers and tags known in the art that readily find use with the RNA replicase polypeptides described herein.
- the RNA replicases having appended sequences that program the RNA replicase molecule for extracellular transport, so that the RNA replicase is secreted to the cell culture medium from the host cell.
- a signal sequence specific for prokaryotic export or eukaryotic export can alternatively be used depending on the application. Any of a variety of signal sequences are available and can be used, as commonly recognized by one of skill in the art. It is not intended that the RNA replicase polypeptides be limited to the particular signal sequences described herein.
- the RNA replicase can be designed with a deletion of the transmembrane domain that is present in the parent polypeptide form (e.g., an RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV)) from which it was derived to achieve improved solubility during protein purification, without interfering with the RNA amplification activity of the modified RNA replicase.
- the RNA replicases of the present disclosure that are designed with a deletion of the transmembrane domain for improved solubility (see, for example, FIG. 3 and SEQ ID NO: 3). Any of the RNA replicases described herein can be similarly designed.
- any combination, any type and any quantity of appended (i.e., fused) amino acid sequences can be used to produce the RNA replicase polypeptides as described herein.
- these recombinant RNA replicase polypeptides can include one or any number of operably fused polypeptide tags, for example, the HIS-tag for ease of detection and purification, one or any number of fused marker protein, for example, a red fluorescent protein, green fluorescent protein, blue fluorescent protein, or any other kind of fluorescent protein marker.
- Other types of beneficial fused sequences that encode polypeptides, or even singe amino acids, can also be operably linked to the RNA replicase polypeptides to produce proteins with additional functionalities.
- these include but are not limited to, signal sequences for post-translational modifications that may be beneficial for the use of the RNA replicase for purposes of the methods described herein, and unnatural amino acids that have beneficial functionalities such as resistance to degradation (improved half-life of the fusion protein) and chemically reactive side chains.
- derivatives or fusions of the RNA replicase molecules provided herein are polypeptides that have been altered so as to exhibit additional features or activities not found on the parent polypeptide form (e.g., an RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV)) from which it was derived.
- RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV)
- Examples include fusion proteins, for example, fusions resulting the RNA replicase molecule also carrying an enzymatic activity to allow detection, for example, a luciferase activity or an alkaline phosphatase activity.
- the precise site at which the fusion is made to produce the fusion polypeptide or protein may be selected empirically to optimize the secretion or binding characteristics of the fusion protein. DNA encoding the fusion protein is then transfected into a host cell for expression.
- nucleic acid sequences including recombinant nucleic acid sequences, encoding an RNA replicase (RNA-dependent RNA polymerase), in particular, an RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV), as described herein.
- RNA replicase RNA-dependent RNA polymerase
- NS5B nonstructural protein 5B
- HCV hepatitis C virus
- the nucleic acid sequence and polynucleotide is operably coupled to at least one regulatory polynucleotide capable of inducing the expression of the RNA replicase in a host cell.
- the host cells comprising the polynucleotide are also a feature of the compositions and methods of the disclosure.
- the present disclosure provides polynucleotide sequences encoding the RNA replicase polypeptides described herein, and variants of these sequences.
- Polynucleotide sequences encoding the RNA replicases described in the present disclosure are provided in FIGS. 2 and 4 and SEQ ID NOS: 2 and 4. It is not intended that polypeptides finding use with the methods described herein be limited to the polynucleotides provided in the present disclosure, as one of skill will recognize that many polynucleotides can be generated that encode any one particular polypeptide, and it is well within the skill of one with ordinary skill to generate alternative polynucleotide sequences encoding the RNA replicases described herein.
- a polynucleotide encoding the RNA replicases described herein can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
- a polynucleotide encoding an RNA replicase described herein can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
- a polynucleotide encoding an RNA replicase described herein can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
- a polynucleotide encoding an RNA replicase described herein may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.
- the polynucleotides may be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of the RNA replicase is known, a polynucleotide encoding the RNA replicase may be assembled from chemically synthesized oligonucleotide. Alternatively, a polynucleotide encoding an RNA replicase may be generated from nucleic acid from a suitable source.
- a nucleic acid encoding the RNA replicase may be chemically synthesized or obtained from a suitable source by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the RNA replicase. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any suitable method.
- the present disclosure provides vector compositions which contain polynucleotide sequences described herein.
- Polynucleotides described herein can be incorporated into any desired DNA or RNA based vector, without limitation.
- a polynucleotide encoding an RNA replicase can be cloned into a suitable expression vector, a subcloning vector, a shuttle vector, a vector designed for use with in vitro transcription reactions, cosmids, phagemids, and vectors derived from mammalian viruses, including retroviruses (for example, lentiviruses), adenoviruses, and adeno-associated viruses (AAV).
- retroviruses for example, lentiviruses
- AAV adeno-associated viruses
- vectors can be conveniently supplied and used in their circular form, or in a linearized form.
- the linearized form finds use in subcloning steps such as in cloning the guide RNA target sequence into the host vector.
- Linearized vectors are also used in in vitro transcription reactions.
- an expression vector can be optimized to express an mRNA in any suitable cell type, for example, bacterial cells, mammalian cells, human cells or mouse cells.
- An expression vector as used herein can be optimally designed to express a protein, e.g., an RNA replicase, in a mammalian host cell.
- a vector comprising protein-coding open reading frame and suitable regulatory elements can be delivered into the host cell by any suitable method of transfection or transduction.
- that ORF is transcribed by endogenous RNA polymerases (RNA pol II in the case where a protein coding gene is expressed) to produce mRNA, and that in turn is translated to produce the encoded protein.
- vector or "expression vector” is used herein to mean vectors used as a vehicle for introducing into and expressing a desired gene in a host cell.
- vectors are readily available, for example, selected from the group consisting of plasmids, phages, viruses and retroviruses.
- vectors can comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.
- numerous expression vector systems may be employed.
- one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper.
- biocide resistance e.g., antibiotics
- the selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals.
- the expression vector may be introduced into an appropriate host cell.
- Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection including lipotransfection using, e.g., Fugene or lipofectamine, protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus.
- transfection including lipotransfection using, e.g., Fugene or lipofectamine, protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus.
- plasmid introduction into the host is via standard calcium phosphate co-precipitation method.
- the host cells harboring the expression construct are grown under conditions appropriate to the production of the RNA replicase. Promoters and Regulatory Elements
- Polynucleotides of the present disclosure are most typically in the context of a vector, such as an expression vector.
- the expression vector can be associated with any type and any quantity of regulatory elements. Such elements can promote the strong and accurate transcription of the polynucleotide, for example, to generate mRNA, either in a cell, or in an in vitro transcription reaction.
- regulatory elements can refer to DNA elements that regulate transcription or regulate translation. Regulatory elements can reside upstream of an open reading frame, or downstream of an open reading frame. It is not intended that the compositions and methods described herein be limited to any particular type or sequence of regulatory elements.
- the polynucleotides of the present disclosure are transcribed within mammalian cells to produce mRNA.
- RNA pol II RNA type II polymerase
- transcription is directed by an RNA type II polymerase (RNA pol II), which requires a mammalian pol II promoter sequence.
- RNA pol II promoter that is used is not limited in any respect. Promoters that function constitutively and promoters that are regulated by induction or repression all find use with the compositions and methods described herein.
- compositions and methods described herein be limited to any of the promoters disclosed herein, as one of ordinary skill in the art recognizes that a wide variety of promoters find are available, for example, constitutively active promoters, inducible promoters, repressible promoters, tissue specific promoters and cell-type specific promoters.
- RNA pol II promoters that can drive high levels of protein expression in mammalian cells may be preferred, and most preferably, are active in a wide range of cell types and species.
- Constitutively active promoters commonly used in mammalian systems include the simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), human ubiquitin C promoter (UBC), human elongation factor 1 alpha promoter and hybrid promoter (EFl-a), mouse phosphogly cerate kinase 1 promoter (PGK), and chicken b-actin promoter coupled with CMV early enhancer (CAG) promoter and murine stem cell virus (MSCV) promoter.
- SV40 simian virus 40 early promoter
- CMV cytomegalovirus immediate-early promoter
- UBC human ubiquitin C promoter
- EFl-a human elongation factor 1 alpha promoter and hybrid promoter
- PGK mouse phosphogly ce
- polypeptide of interest it is desirable to the polypeptide of interest only in certain cell types.
- tissue specific or cell type specific mammalian promoters can be used, which are knows in the art.
- An expression vector of encompassed by the present disclosure can include translation regulatory elements that control accurate translation of the mRNA to produce protein.
- a polypeptide ORF can be linked to translation initiation sequences, translation termination sequences and/or other post-transcriptional regulatory sequences, such as the woodchuck virus post- transcriptional regulatory element (WPRE) to boost gene expression and stabilize the mRNA transcript Host Cells
- WPRE woodchuck virus post- transcriptional regulatory element
- vectors are located within host cells, and host cells are used to express the polypeptides of interest.
- Host cells comprising one or more polynucleotide or vector of disclosure is also provided by the disclosure.
- the polynucleotide that is contained in the host cell can be of any type, for example, an expression vector or a mRNA molecule that was produced by in vitro transcription, or an mRNA molecule that was produced by in vivo transcription within the host cell using endogenous RNA polymerases.
- the type of host cells finding use with the compositions and methods described herein are not limited, as many types of host cells can be used.
- bacterial host cells find use, for example, in propagating and producing plasmid DNA, or producing desired polypeptide.
- Mammalian host cells such as mouse or human cells also find use for producing polypeptides described herein. It is not intended that the disclosure be limited to the any particular host cells described herein, as the disclosure is widely applicable to cells derived from many species, including all primates, mouse, rat, and any other mammalian species, as well as bacterial host systems.
- Stable mammalian cell lines created using the host cells of the disclosure find use for producing polypeptides of interest, namely, the RNA replicases described herein.
- compositions and methods described herein includes host cells comprising a polynucleotide encoding an RNA replicase, which preferably are operably linked to a heterologous promoter.
- host cells refers to cells which harbor vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene.
- cells and “cell culture” are used interchangeably to denote the source of RNA replicase unless it is clearly specified otherwise.
- recovery of polypeptide from the "cells” may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
- the host cell line used for protein expression is often of mammalian origin; those skilled in the art are credited with ability to preferentially determine particular host cell lines which are best suited for the desired gene product to be expressed therein.
- Exemplary host cell lines include, but are not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/0 (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-lclBPT (bovine endothelial cells), RAH (human lymphocyte) and
- a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
- Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
- eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. For long-term, high-yield production of recombinant proteins, stable expression is preferred.
- cell lines that stably express the RNA replicase molecule may be engineered.
- RNA replicase molecule in bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the RNA replicase molecule being expressed. For example, when a large quantity of such a protein is to be produced, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
- RNA replicases of the present disclosure can be produced by any method known in the art for the synthesis of engineered and recombinant protein molecules, in particular, by chemical synthesis or preferably by recombinant expression techniques as described herein.
- the polynucleotide encoding the RNA replicase can be inserted into expression systems contained on vectors that can be transfected into standard recombinant host cells.
- host cells A variety of such host cells may be used; for efficient processing, however, mammalian cells are preferred.
- mammalian cell lines useful for this purpose include, but are not limited to, CHO cells, HEK 293 cells, or NSO cells.
- RNA replicase The production of the RNA replicase is then undertaken by culturing the modified recombinant host under culture conditions appropriate for the growth of the host cells and the expression of the coding sequences. The RNA replicase is then recovered by isolating them from the culture.
- the expression systems can be designed to include signal peptides so that the resulting recombinant protein are secreted into the medium; however, intracellular production is also possible.
- the polynucleotides encoding the RNA replicases are typically inserted in an expression vector for introduction into host cells that may be used to produce the desired quantity of the RNA replicases. Recombinant expression of an RNA replicase is described herein.
- the vector for the production of the RNA replicase molecule may be produced by recombinant DNA technology using techniques well known in the art.
- methods for preparing a protein by expressing a polynucleotide containing an RNA replicase encoding nucleotide sequence are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors containing an RNA replicase coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
- the disclosure provides replication-competent vectors comprising a nucleotide sequence encoding an RNA replicase, operably linked to a promoter.
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Abstract
The present disclosure is directed to compositions, methods, kits, amino acid and nucleic acid sequences, plasmids, vectors, and host cells for direct amplification of target RNA molecules using an RNA replicase, also known as an RNA-dependent RNA polymerase (RdRP), and detection of the amplified target RNA molecules, for molecular diagnosis of pathogens in body fluid or tissue samples.
Description
METHOD FOR DIRECT AMPLIFICATION AND DETECTION OF RNA
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to, and benefit of, U.S. Provisional Application No.
63/025,515, filed May 15, 2020, the entire content of which is hereby incorporated by reference.
TECHNICAL FIELD
[0002] The subject matter relates to methods for direct amplification of target RNA molecules using an RNA replicase, also known as an RNA-dependent RNA polymerase (RdRP), and detection of the amplified target RNA molecules, for molecular diagnosis of pathogens in body fluid or tissue samples. Compositions and kits for use in the direct amplification and detection of target RNA molecules for molecular diagnosis of pathogens, are also described herein.
BACKGROUND
[0003] PCR-based assays for detection of various pathogens, particularly viruses, bacteria and parasites, in clinical samples offer the advantages of high sensitivity and reproducibility, and can be carried out much more rapidly than traditional culturing methods, which can take multiple days. In many cases, rapid analysis is essential in order to properly treat infected individuals and, if necessary, implement procedures to prevent further transmission of infection. Pathogens of significance, which are discussed further below, include various eukaryotic, prokaryotic, and viral pathogens, in particular, viral, bacterial and/or parasitic infections. PCR-based molecular testing allows for sensitive detection of these and other pathogens in patient specimens, in less time than culture testing. However, most PCR protocols nonetheless employ multiple preparation steps, requires testing facilities to have access to a thermocyler, can be time-consuming, and stringent precautions may be needed to avoid contamination of samples.
[0004] In the medical field, ribonucleic acid (RNA) amplification plays an important role in the detection of viral infections in patient material. Worldwide, 80% of all viral infections are caused by RNA viruses, i.e., viral pathogens with a genetic material consisting of RNA. RNA amplification can also play an important role in the detection of various eukaryotic and prokaryotic pathogens, in particular, bacterial and/or parasitic infections. To date, typically RNA has been detected with PCR- based assays by first generating by first generating a complimentary DNA (cDNA) strand of the target RNA with an RNA-dependent DNA polymerase (RdDP), also known as a reverse transcriptase. An additional DNA-dependent DNA polymerase (DdDP) may also be required for more efficient amplification of the cDNA target if the DNA polymerase activity of the RdDP is insufficient to meet assay sensitivity and/or specificity requirements. There is a need for more efficient and faster method of direct amplification of target RNA (i.e., without formation of cDNA or DNA), which can be essentially performed under isothermal conditions (i.e., without requiring thermocycling), and is an
attractive feature for diagnostic tests for identification of pathogenic viral, eukaryotic, and prokaryotic single and double-stranded RNA in a sample.
BRIEF SUMMARY
[0005] The following aspects and embodiments thereof described and illustrated below are meant to be exemplary and illustrative, not limiting in scope.
[0006] In one aspect, a method for detecting a target RNA in a sample is provided. The method comprises (a) adding the sample to an amplification mixture comprising: (i) an RNA replicase (RNA- dependent RNA polymerase); (ii) at least one target oligonucleotide primer specific for the target RNA; (iii) nucleoside triphosphates (NTPs) ATP, CTP, GTP and UTP; (iv) at least one or more of a detectable, target probe for detection of the target RNA; and (v) optionally, an RNAse inhibitor, one or more of a reaction helper protein, or combinations thereof; (b) producing at least one or more of an RNA amplicon using RNA replicase-dependent amplification (RDA) of the target RNA, if present in the sample; and (c) hybridizing the at least one or more of the target probe to the at least one or more of the RNA amplicon to detect presence or absence of the target RNA.
[0007] In an embodiment, the amplification mixture further comprises a control oligonucleotide primer pair specific to a process control sequence and a control probe for detection of the process control sequence.
[0008] In an embodiment, the at least one target oligonucleotide primer and control oligonucleotide primer pair are designed such that amplification and detection of the target RNA and the process control sequence can be performed simultaneously using the same amplification conditions.
[0009] In an embodiment, at least one or more components of the amplification mixture is provided in a form selected from a solid, solution, lyophilized, frozen liquid form, and any combinations thereof.
[0010] In an embodiment, the target RNA is selected from a viral RNA, an mRNA, and a non coding RNA.
[0011] In an embodiment, the non-coding RNA is selected from rRNA, tRNA, and snRNA.
[0012] In an embodiment, the target RNA is a single-stranded RNA (ssRNA), a double- stranded RNA (dsRNA), or combinations thereof.
[0013] In an embodiment, the at least one or more of the RNA amplicon is a single-stranded
RNA amplicon (ssRNA amplicon), a double-stranded RNA amplicon (dsRNA amplicon), or combinations thereof.
[0014] In an embodiment, the at least one or more of the target probe is a single-strand probe that hybridizes to the ssRNA amplicon, one strand of the dsRNA amplicon, or both strands of the dsRNA amplicon.
[0015] In an embodiment, the at least one or more of the target probe is an optically-labeled probe.
[0016] In an embodiment, the optically-labeled probe comprises a fluorescent compound.
[0017] In an embodiment, the amplification is real-time amplification and producing the at least one or more of the RNA amplicon is determined real-time.
[0018] In an embodiment, producing the at least one or more of the RNA amplicon is determined by an end-point analysis.
[0019] In an embodiment, the end-point analysis is achieved by a lateral-flow device.
[0020] In an embodiment, the RDA is performed essentially isothermally.
[0021] In an embodiment, the RDA is performed between 20°C and 75°C.
[0022] In an embodiment, the RDA is performed between 35°C and 70°C.
[0023] In an embodiment, the amplification mixture comprises a single target oligonucleotide primer.
[0024] In an embodiment, the amplification mixture comprises a first target oligonucleotide primer and a second target oligonucleotide primer.
[0025] In an embodiment, the first and second target oligonucleotide primers are provided at equal concentrations.
[0026] In an embodiment, the first and second target oligonucleotide primers are provided at unequal concentrations.
[0027] In an embodiment, the first target oligonucleotide primer is provided at a concentration of at least three times greater than the concentration of the second target oligonucleotide primer. [0028] In an embodiment, the second target oligonucleotide primer is provided at concentration within a range of about 25 nM to about 5 mM.
[0029] In an embodiment, the target RNA is a double-stranded RNA, and wherein the first and second target oligonucleotide primers hybridize to opposite strands of the double-stranded target RNA in 5’ to 3’ orientation.
[0030] In an embodiment, the one or more of the reaction helper protein is a protein that assists in activity of the RNA replicase (RNA-dependent RNA polymerase), stability of the target RNA, separating double-stranded RNA into single-stranded RNA, and combinations thereof.
[0031] In an embodiment, the one or more of the reaction helper protein is an RNA chaperone, an RNA helicase, an RNA binding-factor, a scaffold protein, an RNA modifying enzyme, other types of RNA-binding protein, and combinations thereof.
[0032] In an embodiment, the RNA replicase is a recombinant RNA replicase (RNA- dependent RNA polymerase).
[0033] In an embodiment, the recombinant RNA replicase (RNA-dependent RNA polymerase) is derived from nonstructural protein 5B (NS5B) of hepatitis C virus (HCV).
[0034] In an embodiment, the recombinant RNA replicase (RNA-dependent RNA polymerase) comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
[0035] In an embodiment, presence of the at least one or more of the RNA amplicons is diagnostic for a pathogen or a mutation in a genomic RNA.
[0036] In an embodiment, the pathogen is a eukaryotic pathogen, a prokaryotic pathogen, or a virus.
[0037] In an embodiment, the virus uses cognate RNA replicases (RNA-dependent RNA polymerases) for replication.
[0038] In an embodiment, the virus is selected from a positive-strand genomic RNA virus, a negative-strand genomic RNA virus, and a double-strand genomic RNA virus.
[0039] In another aspect, an in vitro method for detecting an RNA molecule in a sample is provided that comprises combining the sample with an amplification composition comprising (i) an RNA replicase (RNA-dependent RNA polymerase); (ii) at least one oligonucleotide primer specific for the RNA molecule; (iii) nucleoside triphosphates (NTPs) ATP, CTP, GTP and UTP; (iv) at least one or more of a probe comprising a detectable moiety; and (v) optionally, an RNAse inhibitor, one or more of a reaction helper protein, or combinations thereof; generating at least one or more of an RNA amplicon via RNA replicase-dependent amplification (RDA) of the RNA molecule, if present in the sample; and inspecting for presence or absence of signal from the detectable moiety to ascertain presence or absence of the RNA molecule.
[0040] In an embodiment, the at least one or more of the RNA amplicon is generated without formation of a cDNA or DNA.
[0041] In an embodiment, the combining forms a solution of the sample and the amplification composition.
[0042] In an embodiment, the RNA molecule is selected from a viral RNA molecule, an mRNA molecule, and a non-coding RNA molecule.
[0043] In an embodiment, the non-coding RNA molecule is selected from an rRNA, a tRNA, and an snRNA molecule.
[0044] In an embodiment, the target RNA is a single-stranded RNA (ssRNA), a double- stranded RNA (dsRNA), or combinations thereof.
[0045] In an embodiment, the at least one or more of the RNA amplicon is a single-stranded
RNA amplicon (ssRNA amplicon), a double-stranded RNA amplicon (dsRNA amplicon), or combinations thereof.
[0046] In an embodiment, the at least one or more of the probe is a single-strand probe that hybridizes to the ssRNA amplicon, one strand of the dsRNA amplicon, or both strands of the dsRNA amplicon.
[0047] In an embodiment, the probe is an optically-labeled probe.
[0048] In an embodiment, the optically-labeled probe comprises a fluorescent compound.
[0049] In an embodiment, the amplification is real-time amplification and producing the at least one or more of the RNA amplicon is determined real-time.
[0050] In an embodiment, producing the at least one or more of the RNA amplicon is determined by an end-point analysis.
[0051] In an embodiment, the end-point analysis is achieved by a lateral-flow device.
[0052] In an embodiment, the RDA is performed essentially isothermally.
[0053] In an embodiment, the RDA is performed between 20°C and 75°C or between 30°C and 65°C.
[0054] In an embodiment, the amplification composition comprises a single oligonucleotide primer.
[0055] In an embodiment, the amplification composition comprises a first oligonucleotide primer and a second oligonucleotide primer.
[0056] In an embodiment, the first and second oligonucleotide primers are provided at equal concentrations.
[0057] In an embodiment, the first and second oligonucleotide primers are provided at unequal concentrations.
[0058] In an embodiment, the first target oligonucleotide primer is provided at a concentration of at least three times greater than the concentration of the second target oligonucleotide primer. [0059] In an embodiment, the second oligonucleotide primer is provided at concentration within a range of about 25 nM to about 5mM.
[0060] In an embodiment, the RNA molecule is a double-stranded RNA molecule, and wherein the first and second oligonucleotide primers hybridize to opposite strands of the double- stranded RNA molecule in 5’ to 3’ orientation.
[0061] In an embodiment, the one or more of the reaction helper protein is a protein that assists in activity of the RNA replicase (RNA-dependent RNA polymerase), stability of the RNA molecule, separating double-stranded RNA into single-stranded RNA, and combinations thereof.
[0062] In an embodiment, the one or more of the reaction helper protein is an RNA chaperone, an RNA helicase, an RNA binding-factor, a scaffold protein, an RNA modifying enzyme, other types of RNA-binding protein, and combinations thereof.
[0063] In an embodiment, the RNA replicase is a recombinant RNA replicase.
[0064] In an embodiment, the recombinant RNA replicase is derived from nonstructural protein 5B (NS5B) of hepatitis C virus (HCV).
[0065] In an embodiment, the recombinant RNA replicase comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
[0066] In an embodiment, presence of the least one or more of the RNA amplicon is diagnostic for a pathogen or a mutation in a genomic RNA.
[0067] In an embodiment, the pathogen is a eukaryotic pathogen, a prokaryotic pathogen, or a virus.
[0068] In an embodiment, the virus uses cognate RNA-dependent RNA polymerases for replication.
[0069] In an embodiment, the virus is selected from a positive-strand genomic RNA virus, a negative-strand genomic RNA virus, and a double-strand genomic RNA virus.
[0070] In another aspect, a composition for detecting a target RNA in a sample is provided that comprises (i) an RNA replicase (RNA-dependent RNA polymerase); (ii) at least one oligonucleotide primer specific for the target RNA; (iii) nucleoside triphosphates (NTPs) ATP, CTP, GTP and UTP; (iv) at least one or more of a detectable, single-strand probe that hybridizes to the target RNA and an RNA amplicon thereof; and (v) optionally, an RNAse inhibitor, one or more of a reaction helper protein, or combinations thereof. The composition when combined with a sample is capable of producing a single-stranded RNA amplicon (ssRNA amplicon), a double-stranded RNA amplicon (dsRNA amplicon), or combinations thereof, using an isothermal RNA replicase-dependent amplification (RDA) of the target RNA, if present in the sample.
[0071] In an embodiment, the RNA replicase is a recombinant RNA replicase derived from nonstructural protein 5B (NS5B) of hepatitis C virus (HCV).
[0072] In an embodiment, the recombinant RNA replicase comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
[0073] In another aspect, a kit comprising a first container containing a composition as described herein is provided.
[0074] In an embodiment, the kit comprises a second container comprising a master mix, a third container comprising a process buffer, a fourth container containing process control, or any combinations thereof.
[0075] In another embodiment, compositions of one or more containers are provided in lyophilized form, frozen liquid form, or combinations thereof.
[0076] In still another embodiment, the kit further comprises a fifth container containing a rehydration solution.
[0077] In an embodiment, the rehydration solution comprises one or more multivalent cation cofactors for catalyzing enzymes and one or more components for maintaining an optimal pH buffer for an efficient amplification reaction.
[0078] In another aspect, a recombinant RNA replicase (RNA-dependent RNA polymerase) comprising the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto is provided.
[0079] In an embodiment, the recombinant RNA replicase comprises a polypeptide tag.
[0080] In an embodiment, the polypeptide tag is a hexa-histidine peptide (HIS) tag.
[0081] In an embodiment, the recombinant RNA replicase comprises a deletion in the transmembrane domain.
[0082] In another embodiment, a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 4, or an nulciec acid sequence having at least 80%, 90%, or 95% sequence identity thereto is provided
[0083] In another aspect, a plasmid or vector comprising a nucleic acid disclosed herein is provided.
[0084] In another aspect, a host cell comprising disclosed herein is provided.
[0085] In another aspect, a method of generating the recombinant RNA replicase disclosed herein is provided, the method comprising (i) culturing the host cell of claim 80 under conditions sufficient for the expression of the recombinant RNA replicase, and (ii) obtaining the recombinant RNA replicase from the culture.
[0086] Also provided herein are exemplary amino acid and nucleic acid sequences, plasmids, vectors, and host cells useful in embodiments of the methods, compositions, and kits described herein. These include those disclosed in the tables herein.
[0087] These and other objects, features, and embodiments of the present methods, compounds, compositions, kits, plasmids, vectors, host cells and the like, will become more fully apparent from a review of the following detailed description. As can be appreciated from the foregoing and following description, each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present disclosure provided that the features included in such a combination are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment of the present disclosure. Additional aspects and advantages of the present disclosure are also set forth in the following detailed description and claims, particularly when considered in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF FIGURES
[0088] Aspects, features, and advantages of the compositions, methods, kits, amino acid and nucleic acid sequences, plasmids, vectors, host cells and the like described herein, will be understood and more readily apparent when the description in the disclosure is considered in light of the following drawings. It should be appreciated that the drawings are not intended to limit the scope of the present teachings in any way.
[0089] FIG. 1 provides the amino acid sequence of a wild-type hepatitis C virus (HCV) nonstructural 5B protein (NS5B) RNA-dependent RNA polymerase (RNA replicase), which comprises the amino acid residues 2420 to 3010 of Uniprot P26663, herein identified as SEQ ID NO: 1
[0090] FIG. 2 provides the nucleic acid sequence encoding a wild-type hepatitis C virus
(HCV) nonstructural 5B protein (NS5B) RNA-dependent RNA polymerase (RNA replicase), herein identified as SEQ ID NO: 2.
[0091] FIG. 3 provides the amino acid sequence of a recombinant hepatitis C virus (HCV) nonstructural 5B protein (NS5B) RNA-dependent RNA polymerase (RNA replicase), herein identified as SEQ ID NO: 3.
[0092] FIG. 4 provides the nucleic acid sequence encoding a recombinant hepatitis C virus
(HCV) nonstructural 5B protein (NS5B) RNA-dependent RNA polymerase (RNA replicase), herein identified as SEQ ID NO: 4.
BRIEF DESCRIPTION OF THE SEQUENCES [0093] SEQ ID NO: 1: Amino acid sequence of a wild-type hepatitis C virus (HCV) nonstructural 5B protein (NS5B) RNA-dependent RNA polymerase (RNA replicase), which comprises the amino acid residues 2420 to 3010 of Uniprot P26663.
SMSYTWTGAL ITPCAAEESK LPINALSNSL LRHHNMVYAT TSRSAGLRQK KVTFDRLQVL 60
DDHYRDVLKE MKAKASTVKA KLLSVEEACK LTPPHSAKSK FGYGAKDVRN LSSKAVNHIH 120 SVWKDLLEDT VTPIDTTIMA KNEVFCVQPE KGGRKPARLI VFPDLGVRVC EKMALYDWS 180 TLPQWMGSS YGFQYSPGQR VEFLVTWKS KKNPMGFSYD TRCFDSTVTE NDIRVEESIY 240 QCCDLAPEAR QAIKSLTERL YIGGPLTNSK
GQNCGYRRCR ASGVLTTSCG NTLTCYLKAS 300 AACRAAKLQD CTMLVNGDDL W ICESAGTQ EDAASLRVFT
EAMTRYSAPP GDPPQPEYDL 360 ELITSCSSNV SVAHDASGKR VYYLTRDPTT PLARAAWETA RHTPVNSWLG
NIIMYAPTLW 420 ARMILMTHFF SILLAQEQLE KALDCQIYGA CYSIEPLDLP QIIERLHGLS AFSLHSYSPG
480 EINRVASCLR KLGVPPLRVW RHRARSVRAR LLSQGGRAAT CGKYLFNWAV KTKLKLTPIP 540 AASRLDLSGW FVAGYSGGDI YHSLSRARPR WFMLCLLLLS VGVGIYLLPN R 591
[0094] SEQ ID NO: 2: Nucleic acid (DNA) sequence encoding a wild-type hepatitis C virus
(HCV) nonstructural 5B protein (NS5B) RNA-dependent RNA polymerase (RNA replicase). tcaatgtcct acacatggac aggcgccttg atcacgccat gcgctgcgga ggaaagcaag 60
ctgcccatca acgcgttgag caactctttg ctgcgccacc ataacatggt ttatgccaca 120 acatctcgca gcgcaggcct gcggcagaag aaggtcacct ttgacagact gcaagtcctg 180 gacgaccact accgggacgt gctcaaggag atgaaggcga aggcgtccac agttaaggct 240 aaactcctat ccgtagagga agcctgcaag ctgacgcccc cacattcggc caaatccaag 300 tttggctatg gggcaaagga cgtccggaac ctatccagca aggccgttaa ccacatccac 360 tccgtgtgga aggacttgct ggaagacact gtgacaccaa ttgacaccac catcatggca 420 aaaaatgagg ttttctgtgt ccaaccagag aaaggaggcc gtaagccagc ccgccttatc 480 gtattcccag atctgggagt ccgtgtatgc gagaagatgg ccctctatga tgtggtctcc 540 acccttcctc aggtcgtgat gggctcctca tacggattcc agtactctcc tgggcagcga 600 gtcgagttcc tggtgaatac ctggaaatca aagaaaaacc ccatgggctt ttcatatgac 660 actcgctgtt tcgactcaac ggtcaccgag aacgacatcc gtgttgagga gtcaatttac 720 caatgttgtg acttggcccc cgaagccaga caggccataa aatcgctcac agagcggctt 780 tatatcgggg gtcctctgac taattcaaaa gggcagaact gcggttatcg ccggtgccgc 840 gcgagcggcg tgctgacgac tagctgcggt aacaccctca catgttactt gaaggcctct 900 gcagcctgtc gagctgcgaa gctccaggac tgcacgatgc tcgtgaacgg agacgacctc 960 gtcgttatct gtgaaagcgc gggaacccaa gaggacgcgg cgagcctacg agtcttcacg 1020 gaggctatga ctaggtactc cgcccccccc ggggacccgc cccaaccaga atacgacttg 1080 gagctgataa catcatgttc ctccaatgtg tcggtcgccc acgatgcatc aggcaaaagg 1140 gtgtactacc tcacccgtga tcccaccacc cccctagcac gggctgcgtg ggagacagct 1200 agacacactc cagttaactc ctggctaggc aacattatta tgtatgcgcc cactttgtgg 1260 gcaaggatga ttctgatgac tcacttcttc tccatccttc tagcgcagga gcaacttgaa 1320 aaagccctgg actgccagat ctacggggcc tgttactcca ttgagccact tgacctacct 1380 cagatcattg aacgactcca tggccttagc gcattttcac tccatagtta ctctccaggt 1440 gagatcaata gggtggcttc atgcctcagg aaacttgggg taccaccctt gcgagtctgg 1500 agacatcggg ccaggagcgt ccgcgctagg ctactgtccc agggagggag ggccgccact 1560 tgtggcaaat acctcttcaa ctgggcagta aaaaccaaac ttaaactcac tccaatcccg 1620 gctgcgtccc ggctggactt gtccggctgg ttcgttgctg gttacagcgg gggagacata 1680 tatcacagcc tgtctcgtgc ccgaccccgt tggttcatgc tgtgcctact cctactttct 1740 gtaggggtag gcatctacct gctccccaac cgatga 1776
[0095] SEQ ID NO: 3: Amino acid sequence of a recombinant HCV NS5B RNA-dependent
RNA polymerase (RNA replicase).
MHHHHHHSMS YTWTGALITP CAAEESKLPI NALSNSLLRH HNMVYATTSR SAGLRQKKVT 60
FDRLQVLDDH YRDVLKEMKA KASTVKAKLL SVEEACKLTP PHSAKSKFGY GAKDVRNLSS 120 KAVNHIHSVW KDLLEDTVTP IDTTIMAKNE VFCVQPEKGG RKPARLIVFP DLGVRVCEKM 180 ALYDWSTLP QWMGSSYGF QYSPGQRVEF LVNTWKSKKN PMGFSYDTRC FDSTVTENDI 240 RVEESIYQCC DLAPEARQAI KSLTERLYIG GPLTNSKGQN CGYRRCRASG VLTTSCGNTL 300 TCYLKASAAC RAAKLQDCTM LVNGDDLW I CESAGTQEDA ASLRVFTEAM TRYSAPPGDP 360 PQPEYDLELI TSCSSNVSVA HDASGKRVYY LTRDPTTPLA RAAWETARHT PVNSWLGNII 420 MYAPTLWARM ILMTHFFSIL LAQEQLEKAL DCQIYGACYS IEPLDLPQII ERLHGLSAFS 480 LHSYSPGEIN RVASCLRKLG VPPLRVWRHR ARSVRARLLS QGGRAATCGK YLFNWAVKTK 540
LKLTPIPAAS RLDLSGWFVA GYSGGDIYHS LSRARPR 577
[0096] SEQ ID NO: 4: Nucleic acid (DNA) sequence encoding a recombinant HCV NS5B
RNA-dependent RNA polymerase (RNA replicase). catatgcacc accaccacca ccactcaatg agttatacat ggactggagc actgattacc 60 ccgtgcgcgg cggaagaaag caaactgccg atcaacgcgc tgagcaacag cctgctgcgt 120 caccacaaca tggtgtatgc gaccaccagc cgtagcgcgg gtctgcgtca gaagaaagtg 180 accttcgacc gcctgcaagt
tctggacgat cactatcgtg atgttctgaa agagatgaag 240 gcgaaagcga gcaccgtgaa ggcgaaactg ctgagcgttg aggaagcgtg caagctgacc 300 ccgccgcaca gcgcgaagag caaatttggt tacggcgcga aagacgtgcg taacctgagc 360 agcaaggcgg tgaaccacat ccacagcgtt tggaaagacc tgctggagga taccgttacc 420 ccgatcgata ccaccattat ggcgaagaac gaggtgttct gcgttcagcc ggaaaaaggt 480 ggccgtaagc cggcgcgtct gattgtgttt ccggacctgg gcgtgcgtgt ttgcgaaaaa 540 atggcgctgt atgatgtggt tagcaccctg ccgcaagtgg ttatgggtag cagctacggc 600 ttccagtata gcccgggtca acgtgtggag tttctggtta acacctggaa gagcaagaaa 660 aacccgatgg gcttcagcta cgacacccgt tgctttgata gcaccgtgac cgaaaacgac 720 atccgtgttg aggaaagcat ttatcaatgc tgcgatctgg cgccggaggc gcgtcaagcg 780 atcaaaagcc tgaccgaacg tctgtacatt ggtggcccgc tgaccaacag caagggtcag 840 aactgcggct atcgtcgttg ccgtgcgagc ggtgttctga ccaccagctg cggcaacacc 900 ctgacctgct acctgaaagc gagcgcggcg tgccgtgcgg cgaagctgca ggactgcacc 960 atgctggtga acggtgacga tctggtggtt atctgcgaga gcgcgggtac ccaagaagat 1020 gcggcgagcc tgcgtgtttt caccgaagcg atgacccgtt acagcgctcc gccgggtgac 1080 ccgccgcaac cggagtatga tctggaactg attaccagct gcagcagcaa cgtgagcgtt 1140 gcgcacgacg cgagcggcaa acgtgtgtac tatctgaccc gtgatccgac caccccgctg 1200 gcgcgtgcgg cgtgggaaac cgcgcgtcac accccggtta acagctggct gggtaacatc 1260 attatgtacg cgccgaccct gtgggcgcgt atgatcctga tgacccactt ctttagcatt 1320 ctgctggcgc aggagcaact ggaaaaggcg ctggactgcc agatctacgg cgcgtgctat 1380 agcattgagc cgctggatct gccgcaaatc attgaacgtc tgcacggtct gagcgcgttc 1440 agcctgcaca gctacagccc gggcgaaatc aaccgtgtgg cgagctgcct gcgtaaactg 1500 ggtgtgccgc cgctgcgtgt ttggcgtcac cgtgcgcgta gcgttcgtgc gcgtctgctg 1560 agccagggtg gccgtgcggc gacctgcggc aagtacctgt tcaactgggc ggtgaagacc 1620 aaactgaagc tgaccccgat tccggcggcg agccgtctgg acctgagcgg ctggttcgtt 1680 gcgggttata gcggcggcga catttaccat agcctgagcc gtgcgcgtcc gcgttaatga 1740 aagctt 1746
DETAILED DESCRIPTION
[0097] This specification describes various exemplary embodiments of methods, compositions, kits, amino acid and nucleic acid sequences, plasmids, vectors, host cells and the like related to direct amplification of target RNA molecules using an RNA replicase, also known as an RNA-dependent RNA polymerase (RdRP), and detection of the amplified target RNA molecules with various embodiments within the disclosure, for molecular diagnosis of pathogens in body fluid or tissue samples.
[0098] Before the present methods, compositions, kits, amino acid and nucleic acid sequences, plasmids, vectors, host cells and the like are described, it will be understood that this disclosure is not limited to particular embodiments described, and as such may vary. A number of various embodiments of the present disclosure are described in detail hereinafter. These embodiments may take many different forms and should not be construed as limited to those embodiments explicitly set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present disclosure to those skilled in the art. It is also
to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[0099] All patents, applications, published applications and other publications referred to herein, for the purpose of describing and disclosing the compositions, formulations, kits, amino acid and nucleic acid sequences, plasmids, vectors, host cells, and methodologies, are incorporated by reference in their entirety.
I. Definitions
[0100] The terms below, as used herein, have the stated meanings unless indicated otherwise.
Terms and abbreviations not defined should be accorded their ordinary meaning as used in the art. [0101] Note also that singular articles, such as "a" and "an", encompass the plural, unless otherwise specified or apparent from context. For example, reference to a "polymer" includes a single polymer as well as two or more of the same or different polymer, reference to an "excipient" includes a single excipient as well as two or more of the same or different excipients, and the like. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
[0102] The term "ones" means more than one.
[0103] As used herein, the term “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
[0104] As used herein, the terms "comprise", "comprises", "comprising", "contain",
"contains", "containing", "have", "having" "include", "includes", and "including" and their variants are not intended to be limiting, are inclusive or open-ended and do not exclude additional, unrecited additives, components, integers, elements or method steps. For example, a process, method, compound, composition, kit, an amino acid sequence, a nucleic acid sequence, a plasmid, a vector, a host cell and the like that comprises a list of features is not necessarily limited only to those features but may include other features not expressly listed or inherent to such process, method, compound, composition, kit, amino acid sequence, nucleic acid sequence, plasmid, vector, host cell and the like.
[0105] When a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. For example, if a range of 1 mih to 8 mih is stated, it is intended that 2 mih, 3 mih, 4 mih, 5 mih, 6 mih, and 7 mih are also explicitly disclosed, as well as the range of values greater than or equal to 1 mih and the range of values less than or equal to
8 mih. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed by the disclosure. The upper and lower limits of the smaller ranges may be independently included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed by the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range
includes one or both of the limits, ranges excluding either or both of those included limits are also included.
[0106] In some aspects, use of the term “about” indicates that a particular value is intended to include a range of values that represents the limits of accuracy of the instrumentation that was used to obtain the value, or within the range of reproducibility of the value that is being measured by a particular instrument.
[0107] By “specific to” (or “specific for”) a particular pathogen, target pathogen, target, or target sequence (e.g., DNA or RNA sequence) with respect to oligonucleotide primer (e.g., PCR primers or nucleic acid amplification primers) is meant that the primers are substantially complementary, and in some embodiments exactly complementary, to selected primer binding sites in the target DNA or RNA sequence or DNA or RNA sequence of the target pathogen. In some embodiments, the selected primer binding sites are in highly conserved regions of the genome. The sequences of the highly conserved regions may be consensus sequences from sequence alignment of multiple strains of the pathogen. The definition also applies to nucleic acid probes or PCR probes. [0108] By “substantially complementary”, with respect to a PCR primer or probe or a nucleic acid amplification primer or probe, is meant that the oligomer or oligonucleotide primer or probe is sufficiently complementary to its binding site, e.g., in a DNA, cDNA, or an RNA sequence, for efficient binding and amplification to proceed under the conditions of a PCR assay or conditions of other nucleic acid amplification assays, e.g., nucleic acid amplifications conducted under isothermal conditions. In some embodiments, the oligomer or oligonucleotide primer or probe is exactly complementary to its binding site or to a consensus sequence for the binding site, e.g., in a DNA, cDNA, or an RNA sequence. However, there may be one or more mismatches between the primer and/or probe and the binding site in the analyte (e.g., a DNA, cDNA, or RNA sequence) that are tolerated and still result in specific amplification and detection.
[0109] As used herein, "substantially" means sufficient to work for the intended purpose. The term "substantially" thus allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, or the like such as would be expected by a person of ordinary skill in the field but that do not appreciably affect overall performance. When used with respect to numerical values, parameters, or characteristics that can be expressed as numerical values, "substantially" means within five, ten, or twenty percent.
[0110] The term “detection” of a target nucleic acid or analyte, e.g., a DNA, cDNA, an RNA sequence or molecule, refers to determining the presence or the absence of the nucleic acid or analyte in a sample, where absence refers to a zero level or an undetectable level.
[0111] DNA (deoxyribonucleic acid) is a chain of nucleotides consisting of 4 types of nucleotides; A (adenine), T (thymine), C (cytosine), and G (guanine). RNA (ribonucleic acid) is a
chain of nucleotides consisting of 4 types of nucleotides; A (adenine), uracil (U), C (cytosine), and G (guanine). Certain pairs of nucleotides specifically bind to one another in a complementary fashion (called complementary base pairing). That is, adenine (A) pairs with thymine (T) (in the case of RNA, however, adenine (A) pairs with uracil (U)), and cytosine (C) pairs with guanine (G).
[0112] As used herein, the terms “polynucleotide,” “nucleic acid,” "oligonucleotide,"
"oligomer," "oligo” or equivalent terms, refer to molecules that comprises a polymeric arrangement of nucleotide base monomers, where the sequence of monomers defines the polynucleotide. Polynucleotides can include polymers of deoxyribonucleotides to produce deoxyribonucleic acid (DNA), and polymers of ribonucleotides to produce ribonucleic acid (RNA). A polynucleotide can be single- or double-stranded. When single stranded, the polynucleotide can correspond to the sense or antisense strand of a gene. A single-stranded polynucleotide can hybridize with a complementary portion of a target polynucleotide to form a duplex, which can be a homoduplex or a heteroduplex. [0113] The term "polynucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). [0114] Typically, a polynucleotide comprises at least three nucleosides. Usually oligonucleotides range in size from a few monomeric units, e.g., 3-4, to several hundreds of monomeric units. Whenever a polynucleotide such as an oligonucleotide is represented by a sequence of letters, such as “ATGCCTG,” it will be understood that the nucleotides are in 5'->3' order from left to right and that “A” denotes deoxy adenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine, unless otherwise noted. The letters A, C, G, and T (A, C, G, and U in case of an RNA) may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art.
[0115] The term “subject” or “individual” or “animal” or “patient” or “mammal,” as used herein, generally refers to an animal, such as a mammal (e.g., human) or avian (e.g., bird), or other organism, such as a plant. For example, the subject can be a vertebrate, a mammal, a rodent (e.g., a mouse), a primate, a simian, or a human. Animals may include, but are not limited to, farm animals, sport animals, and pets. A subject can be a healthy or asymptomatic individual, an individual that has or is suspected of having a disease (e.g., cancer) or a pre-disposition to the disease, and/or an individual that is in need of therapy or suspected of needing therapy. The term subject includes any subject, particularly a mammalian subject, e.g., a human patient, for whom diagnosis, prognosis, prevention, or therapy is desired. An "isolated" polynucleotide is a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding aa protein, e.g., RNA replicase, contained in a vector is considered isolated. Further examples of an
isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides described herein. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. In addition, polynucleotides may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
[0116] As used herein, it is not intended that the term “polynucleotide” be limited to naturally occurring polynucleotide structures, naturally occurring nucleotides sequences, naturally occurring backbones or naturally occurring intemucleotide linkages. One familiar with the art knows well the wide variety of polynucleotide analogues, unnatural nucleotides, non-natural phosphodiester bond linkages and intemucleotide analogs that find use with the compositions and methods described herein. [0117] As used herein, the expressions “nucleotide sequence,” “sequence of a polynucleotide,”
“nucleic acid sequence,” “polynucleotide sequence”, and equivalent or similar phrases refer to the order of nucleotide monomers in the nucleotide polymer. By convention, a nucleotide sequence is typically written in the 5’ to 3’ direction. Unless otherwise indicated, any particular polynucleotide sequence described herein optionally encompasses complementary sequences, in addition to the sequence explicitly indicated.
[0118] The term “sample,” as used herein, generally refers to a “biological sample” of a subject. The sample can refer to a test sample or a clinical sample, which can include any suitable bodily sample. The sample may be obtained from a tissue or body fluid of a subject. The body fluid can be any of urine, blood, plasma, serum, or any other suitable body fluid. As it pertains to the present disclosure, a body fluid or biological fluid can be a solid, or semi-solid sample, including feces, biopsy specimens, skin, nails, and hair, or a liquid sample, such as urine, saliva, sputum, mucous, blood, blood components such as plasma or serum, amniotic fluid, semen, vaginal secretions, tears, spinal fluid, washings, and other bodily fluids. The sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. The sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample may be a skin sample. The sample may be a cheek swab. The sample may be a plasma or serum sample. Included among the sample are swab specimens from, e.g., the cervix, urethra, nostril, and throat. Any of such samples may be from a living, dead, or dying animal or a plant. Animals include mammals, such as humans.
[0119] The sample may be a cell sample. A cell may be a live cell. The sample may be a cell line or cell culture sample. The sample can include one or more cells. The sample can include one or more microbes or pathogens. The biological sample may be a nucleic acid sample, e.g., DNA or RNA. The sample may be derived from another sample. The sample may be a cell-free or cell free sample. A cell-free sample may include extracellular polynucleotides, e.g., DNA or RNA. Extracellular
polynucleotides may be isolated from a bodily sample that may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool, tears, or any other suitable bodily sample. In some embodiments, the term “sample” can refer to a cell or nuclei suspension extracted from a single biological source (blood, tissue, etc.).
[0120] The sample may comprise any number of macromolecules, for example, cellular macromolecules. The sample maybe or may include one or more constituents of a cell, but may not include other constituents of the cell. An example of such cellular constituents is a nucleus or an organelle. The sample may be or may include DNA, RNA, organelles, proteins, or any combination thereof. The sample may be or include a chromosome or other portion of a genome. The sample may be a cell or one or more constituents from a cell, such as DNA, RNA, nucleus, organelles, proteins, or any combination thereof, from the cell.
[0121] The term “amplicon,” as used herein, generally refers to a piece of a genetic fragment or nucleic acid sequence, e.g., a DNA or an RNA, which can be the source and/or product of amplification or replication events. In the context of molecular biology, the term amplicon refers to the product of an amplification reaction, and hence can be used interchangeably with common laboratory terms, such as a "PCR product." Thus, in this context, amplification can refer to the production of one or more copies of a genetic fragment or target sequence of DNA or RNA, specifically the amplicon. An amplicon can be formed artificially, using various methods including polymerase chain reactions (PCR), other nucleic acid amplification methods, e.g., nucleic acid amplifications conducted under isothermal conditions or essentially isothermal conditions, or ligase chain reactions (LCR), or naturally through gene duplication. Various enzymes can generate DNA or RNA amplicons, for example, a DNA polymerase can generate a DNA amplicon, a reverse transcriptase (RT) or RNA-directed DNA polymerase can generate a DNA amplicon (cDNA) of target mRNA molecules, an RNA replicase or RNA-dependent RNA polymerase can generate a RNA amplicon of target RNA molecules, etc.
[0122] The term “reagent” is used interchangeably herein with the term "component",
"constituent", "substance", "element", "ingredient", etc., and refers to one or more such reagents included in the amplification mixtures, amplification compositions, compositions, etc. of the processes, assays, methods, kits, containers and the like disclosed herein for detecting a target, e.g. target RNA or RNA molecule.
[0123] As used herein, the term “gene” generally refers to a combination of polynucleotide elements, that when operatively linked in either a native or recombinant manner, provide some product or function. The term "gene" is to be interpreted broadly, and can encompass mRNA, cDNA, cRNA and genomic DNA forms of a gene. In some uses, the term "gene" encompasses the transcribed
sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region will contain “open reading frames” that encode polypeptides.
[0124] In some uses of the term, a “gene” comprises only the coding sequences ( e.g . , an “open reading frame” or "coding region") necessary for encoding a polypeptide. In some aspects, genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some aspects, the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters. The term "gene" encompasses mRNA, cDNA and genomic forms of a gene. [0125] As used herein, the terms “vector,” “vehicle,” “construct” and “plasmid” are used in reference to any recombinant polynucleotide molecule that can be propagated and used to transfer nucleic acid segment(s) from one organism to another. Vectors generally comprise parts which mediate vector propagation and manipulation (e.g., one or more origin of replication, genes imparting drug or antibiotic resistance, a multiple cloning site, operably linked promoter/enhancer elements which enable the expression of a cloned gene, etc.). Vectors are generally recombinant nucleic acid molecules, often derived from bacteriophages, or plant or animal viruses. Plasmids and cosmids refer to two such recombinant vectors. A “cloning vector” or “shuttle vector” or “subcloning vector” contain operably linked parts that facilitate subcloning steps (e.g., a multiple cloning site containing multiple restriction endonuclease target sequences). A nucleic acid vector can be a linear molecule, or in circular form, depending on type of vector or type of application. Some circular nucleic acid vectors can be intentionally linearized prior to delivery into a cell.
[0126] As used herein, the term “expression vector” refers to a recombinant vector comprising operably linked polynucleotide elements that facilitate and optimize expression of a desired gene (e.g. , a gene that encodes a protein) in a particular host organism (e.g., a bacterial expression vector or mammalian expression vector). Polynucleotide sequences that facilitate gene expression can include, for example, promoters, enhancers, transcription termination sequences, and ribosome binding sites. [0127] As used herein, the term “host cell” refers to any cell that contains a heterologous nucleic acid. The heterologous nucleic acid can be a vector, such as a shuttle vector or an expression vector. In some aspects, the host cell is able to drive the expression of genes that are encoded on the vector. In some aspects, the host cell supports the replication and propagation of the vector. Host cells can be bacterial cells such as E. coli, or mammalian cells (e.g., cultured human cells or mouse cells). When a suitable host cell (such as a suitable mouse cell) is used to create a stably integrated cell line, that cell line can be used to create a complete transgenic organism. Methods (i.e., means) for delivering vectors/constructs or other nucleic acids (such as in vitro transcribed RNA) into host cells such as bacterial cells and mammalian cells are well known to one of ordinary skill in the art, and are not provided in detail herein. Any method for nucleic acid delivery into a host cell finds use
with the compositions and methods described herein. For example, methods for delivering vectors or other nucleic acid molecules into bacterial cells (termed transformation) such as Escherichia coli are routine, and include electroporation methods and transformation of E. coli cells that have been rendered competent by previous treatment with divalent cations such as CaCh. Methods for delivering vectors or other nucleic acid (such as RNA) into mammalian cells in culture (termed transfection) are routine, and a number of transfection methods find use with the compositions and methods described herein. These include but are not limited to calcium phosphate precipitation, electroporation, lipid- based methods (liposomes or lipoplexes) such as Transfectamine® (Life Technologies™) and TransFectin™ (Bio-Rad Laboratories), cationic polymer transfections, for example using DEAE- dextran, direct nucleic acid injection, biolistic particle injection, and viral transduction using engineered viral carriers (termed transduction, using e.g. , engineered herpes simplex virus, adenovirus, adeno-associated virus, vaccinia virus, Sindbis virus), and sonoporation.
[0128] As used herein, a “polypeptide” or “protein” is any polymer of amino acids (natural or unnatural, or a combination thereol), of any length, typically but not exclusively joined by covalent peptide bonds. A polypeptide can be from any source, e.g., a naturally occurring polypeptide, a polypeptide produced by recombinant molecular genetic techniques, a polypeptide from a cell, or a polypeptide produced enzymatically in a cell-free system. A polypeptide can also be produced using chemical (non-enzymatic) synthesis methods. A polypeptide is characterized by the amino acid sequence in the polymer. The term “peptide” typically refers to a small polypeptide, and typically is smaller than a protein. Unless otherwise stated, it is not intended that a polypeptide be limited by possessing or not possessing any particular biological activity.
[0129] The term "polypeptide" is also intended to include the products of post-expression modifications of the naked amino acid sequence, i.e., modification of the nascent polypeptide, including without limitation and not limited to, glycosylation, acetylation, phosphorylation, amidation and derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non- naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, or produced by chemical synthesis.
[0130] As used herein, an "isolated" polypeptide or a fragment, variant, or derivative thereof is a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinant polypeptides and recombinant proteins expressed in host cells are considered isolated for purposes of the present disclosure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
[0131] Polypeptides as described herein includes fragments, derivatives, analogs or variants of the foregoing polypeptides and any combinations thereof as well. The terms "fragment," "variant,"
"derivative" and "analog" include polypeptides having an amino acid sequence sufficiently similar to the amino acid sequence of the natural peptide, or sequences derived from a parent molecule that is also a recombinant polypeptide. In some embodiments, the polypeptide fragments, derivatives, analogs or variants retain or improve upon the biological activity of the parent polypeptide.
[0132] As used herein, the term “recombinant” in reference to a nucleic acid or polypeptide indicates that the material (e.g., a recombinant nucleic acid, gene, polynucleotide, polypeptide, etc.) has been altered by human intervention, e.g., produced by recombinant DNA techniques. Generally, the arrangement of parts of a recombinant molecule is not a native configuration, or the primary sequence of the recombinant polynucleotide or polypeptide has in some way been manipulated. A naturally occurring nucleotide sequence becomes a recombinant polynucleotide if it is removed from the native location from which it originated (e.g., a chromosome), or if it is transcribed from a recombinant DNA construct.
[0133] A gene open reading frame is a recombinant molecule if that nucleotide sequence has been removed from it natural context and cloned into any type of nucleic acid vector (even if that ORF has the same nucleotide sequence as the naturally occurring gene). Protocols and reagents to produce recombinant nucleic acids and polypeptides are well known to one of ordinary skill in the art. In some embodiments, the term “recombinant cell line” refers to any cell line containing a recombinant nucleic acid, that is to say, a nucleic acid that is not native to that host cell.
[0134] As used herein, the terms “heterologous” or “exogenous” as applied to polynucleotides or polypeptides refers to molecules that have been rearranged or artificially supplied to a biological system and are not in a native configuration (e.g., with respect to sequence, genomic position or arrangement of parts) or are not native to that particular biological system. These terms indicate that the relevant material originated from a source other than the naturally occurring source, or refers to molecules having a non-natural configuration, genetic location or arrangement of parts. The terms "exogenous" and "heterologous" are sometimes used interchangeably with "recombinant."
[0135] As used herein, the term “tag” as used in protein tags refers generally to peptide sequences that are genetically fused to other protein open reading frames, thereby producing recombinant fusion proteins. Ideally, the fused tag does not interfere with the native biological activity or function of the larger protein to which it is fused. Protein tags are used for a variety of purposes, for example but not limited to, tags to facilitate purification, detection or visualization of the fusion proteins. Depending on use, the terms “marker,” “reporter” and “tag” may overlap in definition, where the same protein or polypeptide can be used as either a marker, a reporter or a tag in different applications. In some scenarios, a polypeptide may simultaneously function as a reporter and/or a tag and/or a marker, all in the same recombinant gene or protein.
[0136] As used herein, the term “prokaryote” refers to organisms belonging to the Kingdom
Monera (also termed Procarya), generally distinguishable from eukaryotes by their unicellular organization, asexual reproduction by budding or fission, the lack of a membrane-bound nucleus or other membrane-bound organelles, a circular chromosome, the presence of operons, the absence of introns, message capping and poly-A mRNA, a distinguishing ribosomal structure and other biochemical characteristics. Prokaryotes include subkingdoms Eubacteria (“true bacteria”) and Archaea (sometimes termed “archaebacteria”). As used herein, the terms “bacteria” or “bacterial” refer to prokaryotic Eubacteria, and are distinguishable from Archaea, based on a number of well- defined morphological and biochemical criteria.
[0137] As used herein, the term “eukaryote” refers to organisms (typically multicellular organisms) belonging to the Kingdom Eucarya, generally distinguishable from prokaryotes by the presence of a membrane-bound nucleus and other membrane-bound organelles, linear genetic material (i.e., linear chromosomes), the absence of operons, the presence of introns, message capping and poly- A mRNA, a distinguishing ribosomal structure and other biochemical characteristics. As used herein, the terms “mammal” or “mammalian” refer to a group of eukaryotic organisms in the phylum Chordata that are endothermic amniotes distinguishable from other Chordata by a number of physical, physiological and molecular traits. The group mammals includes the orders Rodentia (including mice and rats) and primates (including humans).
[0138] As used herein, the term “encode” refers broadly to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first. The second molecule may have a chemical structure that is different from the chemical nature of the first molecule.
[0139] For example, in some aspects, the term “encode” describes the process of semi conservative DNA replication, where one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase. In other aspects, a DNA molecule can encode an RNA molecule (e.g., by the process of transcription that uses a DNA-dependent RNA polymerase enzyme). Also, an RNA molecule can encode a polypeptide, as in the process of translation. When used to describe the process of translation, the term “encode” also extends to the triplet codon that encodes an amino acid. In some aspects, an RNA molecule can encode a DNA molecule, e.g., by the process of reverse transcription incorporating an RNA-dependent DNA polymerase. In another aspect, a DNA molecule, for example a gene, can encode a polypeptide, where it is understood that “encode” as used in that case incorporates both the processes of transcription and translation.
[0140] As used herein, the term “derived from” refers to a process whereby a first component
(e.g., a first molecule), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second molecule that is different from the first).
[0141] As used herein, the expression “variant” refers to a first composition (e.g., a first molecule), that is related to a second composition (e.g., a second molecule, also termed a “parent” molecule). The variant molecule can be derived from, isolated from, based on or homologous to the parent molecule. The term variant can be used to describe either polynucleotides or polypeptides. [0142] As used herein, a “variant” polypeptide can refer to a sequence variant where the variant molecule has an amino acid sequence that is not identical to the patent molecule, for example, because the variant molecule contains targeted amino acid substitutions. As applied to polypeptides, a variant molecule can, for example, have 100% amino acid sequence identity with the original parent molecule and comprise additional amino acid residues, or alternatively, can have less than 100% amino acid sequence identity with the parent molecule. For example, a variant of a parent amino acid sequence can be a second amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or any number less than 100% identical in amino acid sequence compared to the original parent amino acid sequence. Polypeptide variants also include polypeptides comprising the entire parent polynucleotide, and further comprising additional fused amino acid sequences. Polypeptide variants also include polypeptides that are portions or subsequences of the parent polypeptide, for example, unique subsequences (e.g., as determined by standard sequence comparison and alignment techniques).
[0143] In another aspect, polypeptide variants include amino acid sequences that contain minor, trivial or inconsequential changes relative to the parent sequence. For example, minor, trivial or inconsequential changes include changes to an amino acid sequence that (i) result in substitutions, deletions or insertions that have little or no impact on the biological activity of the polypeptide, and (ii) result in the substitution of an amino acid with a chemically similar amino acid, i.e., conservative amino acid substitutions.
[0144] Polypeptide variants include polypeptides comprising the entire parent polypeptide, and further comprising additional fused amino acid sequences. Polypeptide variants also includes polypeptides that are portions or subsequences of the parent polypeptide, for example, unique subsequences (e.g., as determined by standard sequence comparison and alignment techniques) of the polypeptides disclosed herein are also encompassed by the compositions and methods described herein.
[0145] In another aspect, polypeptide variants includes polypeptides that contain minor, trivial or inconsequential changes to the parent amino acid sequence. For example, minor, trivial or inconsequential changes include amino acid changes (including substitutions, deletions and insertions)
that have little or no impact on the biological activity of the polypeptide, and yield functionally identical polypeptides, including additions of non-functional peptide sequence. In other aspects, the variant polypeptides change the biological activity of the parent molecule. One of skill will appreciate that many variant polypeptides can be derived from the polypeptides provided by the present disclosure.
[0146] Variant polynucleotides are also provided by the present disclosure. In some aspects, variant polynucleotides are any polynucleotide that encodes a variant polypeptide.
[0147] In other aspects, a variant polynucleotides can have entire nucleotide sequence identity with the original parent polynucleotide molecule and contain additional sequence information, or alternatively, can have less than 100% nucleotide sequence identity with the parent molecule. For example, a variant of a parent nucleotide sequence can have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or any value less than 100% identity with the patent nucleotide sequence. Polynucleotide variants also include polynucleotides comprising the entire parent polynucleotide, and further comprising additional fused nucleotide sequences. Polynucleotide variants also includes polynucleotides that are portions or subsequences of the parent polynucleotide, for example, unique subsequences (e.g., as determined by standard sequence comparison and alignment techniques). [0148] In another aspect, polynucleotide variants includes nucleotide sequences that contain minor, trivial or inconsequential changes to the parent nucleotide sequence. For example, minor, trivial or inconsequential changes include changes to nucleotide sequence that (i) do not change the amino acid sequence of the corresponding polypeptide, e.g., substitutions in codon wobble positions, (ii) occur outside the protein-coding open reading frame of a polynucleotide, (iii) result in deletions or insertions that may impact the corresponding amino acid sequence, but have little or no impact on the biological activity of the polypeptide, (iv) the nucleotide changes result in the substitution of an amino acid with a chemically similar amino acid, i.e., conservative amino acid substitutions. In the case where a polynucleotide does not encode for a protein (for example, a tRNA or a crRNA or a tracrRNA), variants of that polynucleotide can include nucleotide changes that do not result in loss of function of the polynucleotide. In another aspect, conservative variants of the disclosed nucleotide sequences that yield functionally identical nucleotide sequences are encompassed by the compositions and methods described herein. One of skill will appreciate that many variant polynucleotides can be derived from the disclosed nucleotide sequences.
[0149] In some aspects, polynucleotide or polypeptide variants can include variant molecules that alter, add or delete a small percentage of the nucleotide or amino acid positions, for example, typically less than about 10%, less than about 5%, less than 4%, less than 2% or less than 1%.
[0150] As used herein, the terms “identical” or “percent identity” in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same
(“identical”) or have a specified percentage of amino acid residues or nucleotides that are identical (“percent identity”) when compared and aligned for maximum correspondence with a second molecule, as measured using a sequence comparison algorithm (e.g., by a BLAST alignment, or any other algorithm known to persons of skill), or alternatively, by visual inspection.
[0151] The phrase “substantially identical,” in the context of two nucleic acids or polypeptides refers to two or more sequences or subsequences that have at least about 60%, about 80%, about 90%, about 90-95%, about 95%, about 98%, about 99% or more nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence using a sequence comparison algorithm or by visual inspection. Such “substantially identical” sequences are typically considered to be “homologous,” without reference to actual ancestry. Preferably, the “substantial identity” between nucleotides exists over a region of the polynucleotide at least about 50 nucleotides in length, at least about 100 nucleotides in length, at least about 200 nucleotides in length, at least about 300 nucleotides in length, or at least about 500 nucleotides in length, most preferably over their entire length of the polynucleotide. Preferably, the “substantial identity” between polypeptides exists over a region of the polypeptide at least about 50 amino acid residues in length, more preferably over a region of at least about 100 amino acid residues, and most preferably, the sequences are substantially identical over their entire length.
[0152] The phrase “sequence similarity,” in the context of two polypeptides refers to the extent of relatedness between two or more sequences or subsequences. Such sequences will typically have some degree of amino acid sequence identity, and in addition, where there exists amino acid non identity, there is some percentage of substitutions within groups of functionally related amino acids. For example, substitution of a serine with a threonine in a polypeptide is sequence similarity, but not identity.
[0153] As used herein, the term “homologous” refers to two or more polypeptides when they are derived, naturally or artificially, from a common ancestral polypeptide. Similarly, nucleotide sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid. Homology in proteins is generally inferred from amino acid sequence identity and sequence similarity between two or more proteins. The precise percentage of identity and/or similarity between sequences that is useful in establishing homology varies with the polynucleotide and polypeptide at issue, but as little as 25% sequence similarity is routinely used to establish homology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used to establish homology. Methods for determining sequence similarity percentages (e.g., BLASTP and BLASTN using default parameters) are generally available and well known to one of skill in the art.
[0154] As used herein, the terms “portion,” “subsequence,” “segment” or “fragment” or similar terms refer to any portion of a larger sequence (e.g., a nucleotide subsequence or an amino acid subsequence) that is smaller than the complete sequence from which it was derived. The minimum length of a subsequence is generally not limited, except that a minimum length may be useful in view of its intended function. The subsequence can be derived from any portion of the parent molecule. In some aspects, the portion or subsequence retains a critical feature or biological activity of the larger molecule, or corresponds to a particular functional domain of the parent molecule, for example, the DNA-binding domain, or the transcriptional activation domain.
II. Assay Reagents and RNA Replicase-Dependent Amplification (RDA)
[0155] In various embodiments, disclosed herein is an assay for direct amplification and qualitative detection of target RNA molecules and sequences, e.g., from various eukaryotic, prokaryotic, and viral pathogens, in test samples, such as patient body fluids or tissue samples. In some embodiments, the assay is a real time assay. In some embodiments, the assay is a multiplex assay that can detect multiple pathogens. In some embodiments, the assay is a multiple, real time assay. In some embodiments, the assay disclosed herein can be also employed for detection of a mutation in the genomic RNA. For example, the assay can be employed for detection of one or more mutations e.g., single-nucleotide polymorphisms (SNPs), in a genomic RNA. In particular embodiments, the multiplex real time assay within the disclosure relates to an RNA replicase- dependent amplification (RDA) of target RNA molecules using an RNA replicase, also known as an RNA-dependent RNA polymerase or RdRP, and detection of the amplified target RNA molecules and sequences for molecular diagnosis of various pathogens in body fluid or tissue samples or a mutation in the genomic RNA. The in vitro diagnostic assay is directed towards the diagnosis of various eukaryotic, prokaryotic, and viral pathogens, in particular, viral, bacterial and/or parasitic infections, in subjects, particularly human subjects and patients, or a mutation in the genomic RNA. In one embodiment, the assay may also provide differential detection of the presence or absence of multiple pathogens in a single assay. In yet another embodiment, the assay may also provide differential detection of the presence or absence of a pathogen by detecting multiple targets to the pathogen in a single assay. Advantageously, the direct RNA amplification assays disclosed herein can be performed in less than 3 hours, in some cases less than 2.5 hours, and in some cases even in less than 75 minutes. [0156] In one embodiment, the reagents employed in the assays are provided in solid form, for example in a lyophilized form. In one embodiment, the reagents employed in the assays are provided in solution form, for example in a frozen liquid form. In some embodiments, the different reagents employed in the assays are provided in various different forms, for example, some reagents are provided in solid forms, for example in a lyophilized form, whereas some other reagents are provided in solution forms, for example in a frozen liquid forms. In some embodiments, the reagents employed
in the assays are provided in a single container. In some embodiments, the reagents employed in the assays are provided in separate containers, for example, in two, three, four, five or more containers. In some embodiments, after sample preparation, the reagents can be simply combined with the liquid sample. In other embodiments, after sample preparation, reagents provided in solid form can be simply rehydrated using a rehydration solution, for example, a rehydration solution provided as a reagent in one of the containers, and combined with the liquid sample. The assay can thus be carried out with a minimal amount of transfer of reagent solutions, greatly reducing the possibility of contamination or loss of sample, as well as the time needed for completion of the assay.
[0157] The reagents and components of the amplification mixtures, amplification compositions, compositions and the like employed herein for direct amplification and detection of a target, e.g. a target RNA or RNA molecule or sequence, as described in more detail below, can include:
(i) an RNA replicase (RNA-dependent RNA polymerase);
(ii) at least one target oligonucleotide primer specific for the target RNA;
(iii) nucleoside triphosphates (NTPs) ATP, CTP, GTP and UTP;
(iv) at least one or more a detectable, target probe for detection of the target RNA; and
(v) optionally, an RNAse inhibitor, one or more of a reaction helper protein, or combinations thereof.
[0158] In various embodiments, and as described further below, the target RNA molecules and sequences that can be amplified and detected by employing the multiplex real time PCR-based assays within the disclosure, in particular, by the RNA replicas e-dependent amplification (RDA) disclosed herein, include, but are not limited to, viral RNAs, mRNAs, and other non-coding RNAs, such as rRNAs, tRNAs, snRNAs, etc.
[0159] In various embodiments, a sample containing a target RNA molecule or sequence can be prepared, as described further below, combined with or added to the amplification mixtures, amplification compositions, and compositions described herein, and RNA amplicons of the target RNA molecule or sequence can be produced using, for example, an RNA replicase-dependent amplification (RDA) of the target RNA molecule or sequence, if and when such target RNA is present in the sample. An amplicon can refer to a piece of a genetic fragment or nucleic acid sequence, e.g., an RNA molecule or sequence, which can be the source and/or product of amplification or replication events. As used herein, an RNA amplicon refers to the product of a RNA amplification reaction, in particular, an RNA replicase-dependent amplification (RDA) of the target RNA molecule or sequence, which within various embodiments of the disclosure can be performed under isothermal conditions. In various embodiments, the RNA amplicons that can be produced by the RDA disclosed herein include single-stranded RNA amplicons (ssRNA amplicons), double-stranded RNA amplicons (dsRNA amplicons), or combinations thereof.
[0160] In some embodiments, the compositions described herein include an enzyme that possesses an RNA polymerase activity for amplification of an RNA molecule or sequence (e.g. a target RNA molecule or sequence from a pathogen). In specific embodiments, the enzyme can be an RNA replicase, also known as an RNA-dependent RNA polymerase or RdRP, which possesses an RNA polymerase activity and is capable of direct amplification of RNA molecule or sequence (e.g. a target RNA molecule or sequence from a pathogen) without formation of a cDNA or DNA. In some embodiments, the RNA replicase enzyme is further capable of performing the amplification of RNA, also referred to as the RNA replicase-dependent amplification (RDA) of RNA, essentially isothermally. In some embodiments, the RNA replicase is derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV), and comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto. In some embodiments, the RNA replicase is a recombinant RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV). In some embodiments, the recombinant RNA replicase comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto. The composition can include one or more stabilizers. In various embodiments, a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1, is disclosed herein. A nucleic acid encoding the amino acid sequence of SEQ ID NO: 3, is also disclosed herein. Exemplary nucleic acid sequences of the RNA replicase of the various embodiments disclosed herein can include the nucleic acid sequence set forth in SEQ ID NO: 2, or a nucleic acid sequence having at least 80%, 90%, or 95% sequence identity thereto, and SEQ ID NO: 4, or a nucleic acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
[0161] Also contained in the compositions described herein are four different nucleoside triphosphates (NTPs): ATP, CTP, GTP and UTP. RNA contains four different nitrogenous bases: adenine (A), guanine (G), and cytosine (C), and uracil (U). RNA synthesis is catalyzed by RNA polymerases that utilizes the NTPs as substrates to synthesize the RNA chain. In some embodiments, the four different NTPs ATP, CTP, GTP and UTP are each present in equal concentrations that is appropriate for the assays disclosed herein. In some embodiments, the four different NTPs ATP, CTP, GTP and UTP are each present in different concentrations that is appropriate for the assays disclosed herein.
[0162] In various embodiments, such reagents may include an RNAse inhibitor (New England
Biolabs, Inc., Ipswich, Mass; RNAsin, Promega) in an appropriate reaction concentration. In various embodiments, the compositions described herein can also include one or more reaction helper proteins. In some embodiments, the reaction helper protein can be a protein that assists in the activity of the RNA replicase (RNA-dependent RNA polymerase), stabilizes the target RNA, or separates a double- stranded RNA into single-stranded RNA. In some embodiments, the reaction helper protein can be an
RNA-binding protein (RBP), which includes, but is not limited to, an RNA chaperone, an RNA helicase, an RNA binding-factor, a scaffold protein, or an RNA-modifying enzyme that may be required for, but not limited to, an RNA replicase activity, target RNA-binding, and/or assisting in stability of a target RNA. In some embodiments, the RNA helicase can be used to separate strands in a duplex (i.e., double-stranded RNA), allowing primers to bind the duplex and be extended during the amplification process.
[0163] It is understood that, the compositions described herein can further include any reagent that is conventionally employed for nucleic acid amplification, more specifically, for direct and/or isothermal RNA amplification utilizing RDA, including those that are not explicitly disclosed herein.
Oligonucleotide Primers
[0164] In various embodiments, exemplary primers are designed, and amplification conditions selected, such that efficient RNA amplification, e.g., RNA replicase-dependent amplification (RDA), and detection of one or more target RNA molecules and sequences can be performed simultaneously using the same amplification conditions. Exemplary primer sets having this property are described below.
[0165] In some embodiments, the compositions described herein include at least one target oligonucleotide primer specific for a target RNA molecule or sequence to be detected (analyte), e.g., an RNA molecule or sequence derived from a pathogen. Typically, the oligonucleotide primer or primers are specific for selected primer binding sites, e.g., in the RNA sequence, which can be in highly conserved regions of a genome, e.g., genome of the pathogen, to be detected.
[0166] In some embodiments, the composition can include a single target oligonucleotide primer that is capable of performing successful asymmetric amplification of RNA. Typically, a single target oligonucleotide primer may be employed for avoiding generation of double-stranded RNA amplicons (dsRNA amplicons), while specifically generating single-stranded RNA amplicons (ssRNA amplicons) that can be advantageous for employing certain RNA amplicon detection methodologies, e.g., real-time and end-point RNA amplicon detection methodologies.
[0167] In some embodiments, the composition can include an oligonucleotide primer pair comprising a first target oligonucleotide primer and a second target oligonucleotide primer. In some embodiments, the first and second target oligonucleotide primers are provided in the composition in equal concentrations. In some embodiments, the first and second target oligonucleotide primers are provided in the composition in unequal concentrations. When the first and second target oligonucleotide primers are provided in unequal concentrations, the first target oligonucleotide primer can be present at a concentration that is at least two times, at least three times, at least four time, or at least five times greater than the concentration of the second target oligonucleotide primer. In some embodiments, the second target oligonucleotide primer is provided at concentration within a range of
about 25 nM to about 5 mM, including at least about 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 75 nM, 100 nM, 150 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 mM, 1.5 mM , 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, etc.
[0168] In some embodiments, the compositions described herein include an oligonucleotide primer pair comprising a first target oligonucleotide primer and a second target oligonucleotide primer that are specific for a double-stranded target RNA to be detected. In such embodiments, the first and second target oligonucleotide primers can hybridize to opposite strands of the double-stranded RNA (dsRNA) in 5’ to 3’ orientation for the amplification process.
[0169] In some embodiments, the composition can further include a control oligonucleotide primer or primer pair specific to a process control sequence, e.g., a process control RNA sequence, and optionally, a control probe for detection of the process control sequence. Typically, the target oligonucleotide primer or primer pair and control oligonucleotide primer or primer pairs are designed such that amplification and detection of the target RNA and the process control sequence, can be performed simultaneously using the same amplification conditions. Accordingly, the primers are designed such that the annealing/melting temperatures of primer/binding site duplexes for one or more target RNA sequences (e.g., analytes) and for the process control sequence, are approximately equivalent, e.g., within 3 °C, within 2 °C, or within 1 °C or less, in the amplification reaction environment. In other embodiments, the annealing/melting temperatures of primer/binding site duplexes for one or more target RNA sequences (e.g., analytes) and for the process control sequence, are within 5 °C, or are nearly equivalent, e.g., within 0.5 °C or less.
[0170] In some embodiments, to detect a target RNA sequence of a pathogen with high sequence variability, e.g., of a viral pathogen with high sequence variation, multiple variations of a primer sequence may be required to adequately detect all possible variations of the viral target RNA sequences. In some embodiments, degenerate bases (such as, R = A/G, Y = C/G, W = A/T, S = C/G, K = G/T, M = A/C) may be used for effective detection of all possible variations of a pathogenic target RNA sequences. Alternatively, in some embodiments, a few different versions of a primer sequence may be used for effective detection of all possible variations of a pathogenic target RNA sequences. In some embodiments, to accommodate annealing temperatures, one primer may be designed to be longer, shorter, or shifted as compared to the other primers for effective detection of all possible variations of a pathogenic target RNA sequences.
[0171] The oligonucleotide primer or oligonucleotide primer pairs of the disclosure are preferably specific to or specific for selected primer binding sites in the genomic target RNA sequence (e.g., for detecting mutations in the genomic RNA) or to an RNA sequence of the target pathogen (e.g., for detecting presence of target pathogen(s) in a sample). In some embodiments, the oligonucleotide primer or oligonucleotide primer pairs of the disclosure are preferably specific to
regions in the genome of the target pathogen(s), in particular, to highly conserved regions in the genome of the target pathogen(s) described in detail below. In some embodiments of the method, the primers are substantially complementary, and in some embodiments exactly complementary, to selected regions of regions, including highly conserved regions, of RNA, such as viral, bacterial or parasitic RNA. The sequences of the highly conserved regions used for primer design may be consensus sequences derived from multiple strains and/or subtypes of a pathogen.
Detection of Amplified Products
[0172] To detect amplified nucleic acid, e.g., RNA amplicons, the primers and probes of various embodiments herein may be subject to various modification, such as fluorescent or chemiluminescent-labeling, and biotinylation. Amplified nucleic acid products, e.g., RNA amplicons, may be detected by various known methods including end-point ethidium-bromide staining, or by detecting the amplified products by means of a label selected from a radiolabel, a fluorescent-label, and an enzyme. Labeling methods may also include radioactive isotopes, chromophores, and ligands such as biotin or haptens, which can be readily detected by reaction with labeled forms of their specific binding partners, e.g., avidin and antibodies respectively. In some embodiments, the probes are RNA probes that include, but are not limited to, FITC, DIG, or DNP labeled probes, which bind to the RNA amplicons, for example, to single-stranded amplicons or to one or both strands of double-stranded amplicons.
[0173] In some embodiments, the compositions described herein can also include specific
RNA probes labeled with a quencher on one end and a fluorophore reporter on the other end for binding to the RNA amplicons, and in some embodiments, to biotinylated RNA amplicons. Upon annealing to the RNA amplicons, the quencher is separated from the fluorophore reporter on the probe, thereby de-quenching the fluorophore and resulting in an increase in the detectable fluorescent signal. In some embodiments, specific RNA probes labeled with a quencher on one end and a fluorophore reporter on the other end for binding to the RNA amplicons may include a hairpin loop between the fluorophore and the quencher. In some embodiments, specific RNA probes labeled with a quencher on one end and a fluorophore reporter on the other end for binding to the RNA amplicons may not include a hairpin loop between the fluorophore and the quencher. In such embodiments, upon annealing, the probe without a hairpin loop may need to be hydrolyzed by a polymerase (e.g., TaqMan) or enzymatically cut in order to un-quench the fluorophore from the quencher. In the embodiments described above, with each amplification cycle, additional fluorophore reporter molecules are separated from their quenchers, resulting in additional signal. If sufficient fluorescence is achieved by a predetermined number of cycles, the sample can be reported as positive for the detected RNA sequence. Accordingly, in some embodiments, the RNA amplification and detection assays within
the disclosure can provide for real-time detection of a target RNA sequence using such fluorescent- labeled probes.
[0174] In some embodiments, the compositions described herein can include primers, e.g., biotinylated primers, which bind to target RNA sequences that have been separated during the amplification process. In some embodiments, the compositions described herein can further include labeled RNA probes, e.g., FITC, DIG, or DNP labeled probes, which bind to the biotinylated RNA amplicons, for example, to the single-stranded biotinylated amplicons or to one or both strands of double-stranded biotinylated amplicons. In such embodiments, the biotinylated amplicons can bind to streptavidin coated beads and allow for end-point detection of the amplicons on surfaces coated with anti-FITC antibodies.
[0175] Accordingly, in various embodiments, the compositions described herein can include a detectable probe for detection of the target RNA, including detection of the RDA amplified RNA amplicons. The detectable probes can hybridize to the RNA target sequence and/or to one or more of the RNA amplicons that are produced during the RNA amplification, e.g., RDA, of the target RNA, if the target RNA is present in the sample, and thereby allows for detection of presence or absence of the target RNA in the sample. In some embodiments, the compositions described herein can also include a control probe for detection of the process control.
[0176] In various embodiments, the detectable probe can be an optically-labeled probe, for example, an optically-labeled probe that includes a fluorescent compound. In some embodiments, as described above, the RNA amplification is a real-time amplification, and the RNA amplicons produced during the amplification is detected real-time. In some embodiments, the RNA amplicons produced during the RNA amplification can be determined by an end-point analysis, for example, an end-point analysis achieved by a lateral-flow using, for example, biotinylated primers, RNA probes, e.g., FITC, DIG, or DNP labeled probes (that bind to the RNA amplicons and biotinylated RNA amplicons), and anti-FITC antibodies, as described above.
Asymmetric RNA Reylicase-Deyendent Amplification (RDA)
[0177] Unequal concentrations of oligonucleotide primers can be used to preferentially generate single-stranded amplicon for hybridization to a probe for detection of a target nucleic acid segment, e.g. an RNA. In various embodiments, asymmetric RDA may be performed isothermally at a temperature in the range of about 20 °C and 75 °C, within a range of 35 °C and 70 °C, or more specifically, within a range of 60 °C and 66°C for a thermophilic RDA reaction. In various embodiments, the first or forward oligonucleotide primer can be provided at a higher concentration, e.g., at least three fold excess, than the second or reverse oligonucleotide primer, which serves as the limiting primer. Alternatively, the second or reverse oligonucleotide primer can be provided in at least three-fold excess where the first or forward oligonucleotide primer serves as the limiting primer.
Depletion of the limiting primer during exponential amplification results in linear synthesis of the product generated from the excess primer. This leads to accumulation of the single-stranded amplicon that originated from the excess primer. In various embodiments, the limiting primer can be provided in the range of about 25 nM to about 5 mM, or in the range of about 25 nM to about 75 nM (e.g., about 50 nM). In various embodiments, the excess primer can be provided at a concentration that is in the range of approximately 1.5 to 10 times that of the limiting primer (for example, 200 nM). In one embodiment, the excess primer can be provided at a concentration that is three or four times higher than the concentration of the limiting primer.
Amplification Conditions
[0178] As noted above, the primers are designed, and amplification conditions selected, such that efficient amplification, e.g., RNA replicase-dependent amplification (RDA), and detection of one or more target RNA molecules and sequences can be performed simultaneously using the same thermal cycling conditions on a thermocycler. In some embodiments, the primers are designed, and amplification conditions are selected, such that efficient amplification, e.g., RNA replicase-dependent amplification (RDA), and detection of one or more target RNA molecules and sequences can be performed simultaneously using the same isothermal conditions, for example in the range of 20 °C and 75 °C, within a range of 35 °C and 70 °C, or within a range of 60 °C and 66°C, and essentially without a thermocycler and under isothermal conditions. Accordingly, a multiplex RNA amplification reaction can be carried out under optimized conditions in a single vessel, or in multiple vessels but under the same RNA amplification conditions, generating amplicons for one or more of the target pathogens present in a sample. Furthermore, as described above, the primers used in assays disclosed herein, are designed such that the annealing/melting temperatures of primer/binding site duplexes for one or more target RNA sequences (e.g., analytes) and for the process control sequence, are approximately equivalent, e.g., within 3 °C, within 2 °C, or within 1 °C or less, in the amplification reaction environment. In other embodiments, the annealing/melting temperatures of primer/binding site duplexes for one or more target RNA sequences (e.g., analytes) and for the process control sequence, are within 5 °C, or are nearly equivalent, e.g., within 0.5 °C or less.
Analytes
[0179] Applications of this method can include detection and diagnosis of a pathogen in a sample (e.g., a clinical or test sample), detecting a mutation in a genomic RNA (e.g., human genomic RNA), and single polynucleotide detection. In some embodiments, the current multiplex and/or real time assays within the disclosure are useful for detection and diagnosis of various pathogens, for example, various eukaryotic, prokaryotic, and viral pathogens, in clinical samples, such as body fluids or tissue samples. In particular, the in vitro diagnostic assays within the disclosure relates to direct
amplification of target RNA molecules (i.e., analytes) using an RNA replicase-dependent amplification (RDA) and detection of the amplified target RNA molecules for molecular detection of a mutation in a genomic RNA, as well as diagnosis of various pathogens, in particular, viral, bacterial and/or parasitic infections, in test samples from subjects, particularly human subjects and patients. [0180] Viral pathogens of significance may include, for example, viruses that use cognate
RNA replicases (RNA-dependent RNA polymerases) for replication. In various embodiments, viral pathogens may include, for example, a positive-strand genomic RNA virus, a negative-strand genomic RNA virus, and a double-strand genomic RNA virus. In particular embodiments, viral pathogens may include, for example, the human immunodeficiency virus (HIV), the hepatitis C virus (HCV), coronavirus, Ebola virus, the hepatitis A virus, the flu viruses (influenza A, B and C), Avian influenza virus, the SARS virus, the polio virus (Poliovirus), the measles virus, the mumps virus, the rubella virus, rotavirus, sapovirus, norovirus, human respiratory syncytial virus (RSV) A and B, human metapneumo virus (hMPV), herpes simplex virus 1 and 2 (HSV-1 and HSV-2), human parainfluenza virus (HPIVs) 1-4, Varicella-zoster virus (VZV, also referred to as HSV-3.
[0181] Pathogens of significance may also include, but are not limited to, Clostridium difficile
(C. diff), various Staphylococcus species, such as methicillin-resistant Staphylococcus aureus (MRSA), healthcare-associated MRSA (HA-MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant coagulase-negative staphylococci (MRCNS), methicillin-sensitive coagulase-negative staphylococci (MSCNS), methicillin-resistant Staphylococcus epidermidis (MRSE) and methicillin-sensitive Staphylococcus epidermidis (MRSE), Group B streptococcus, Bordetella pertussis, Bordetella parapertussis, Bordetella holmesii, and parasites such as, Cryptosporidium species, Entamoeba species including E. histolytica, Giardia lamblia, and Microsporidia. In some embodiments, pathogens may include a mycobacterium species including, but not limited to, Mycobacterium kansasii, Mycobacterium tuberculosis, Mycobacterium intracellualre, Mycobacterium tuberculosis , Mycobacterium avium, and Mycoplasma pneumonia. [0182] In some embodiments, pathogens may include parasitic helminth and protozoan parasites including, but not limited to, Acanthamoeba species, Anisakis species, Ascaris lumbricoides , Botfly, Balantidium coli, Bedbugs, Cestoidea (tapeworms), Chiggers, Cochliomyia hominivorax, Cryptosporidium species, Entamoeba species including E. histolytica, Fasciola hepatica and other liver flukes, Giardia species (e.g., G. lamblia), Hookworm, Leishmania, Linguatula serrata, Loa loa, Microsporidia, Paragonimus, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis and other pinworms, mites, Toxoplasma gondii, Trypanosoma, Whipworm and Wuchereria bancrofti.
Test Samples
[0183] The test sample, simply referred to as sample, may be any clinical sample, e.g., any body fluid or tissue sample including, but not limited to, mammalian blood, serum, plasma, or cells,
suspected of containing a target pathogen, collected according to procedures known in the art. For example, respiratory viruses may be detected in a nasal swab, nasophyrangeal swab, or nasal aspirate/wash specimens. As another example, the target pathogen(s) may be detected from a stool sample. Extraction of nucleic acids, e.g., RNA, from the test sample may be performed manually or automatically, as known in the art, using the appropriate reagents and following the manufacturer's instructions for automated systems. Automated sample extraction platforms include, for example, the NucliSENS® easyMag® system (bioMerieux). In some embodiments, no extraction step is needed or performed.
[0184] In some embodiments, a process control can be added to an aliquot of every specimen prior to the extraction procedure. The process control serves to assure adequate nucleic acid extraction and to reflect the presence of any inhibitors that may be present in the sample.
[0185] In some embodiments, when a solid reagent composition used, such reagent is rehydrated using a rehydration solution, and aliquots are placed in amplification reaction tubes or plate wells. Aliquots of prepared fluid sample, containing nucleic acids and process control, are then added. Alternatively, the rehydrated reagents can be added to the fluid sample. Amplification, e.g., RDA, is then carried out in a thermal cycling apparatus, or without requiring thermocy cling under isothermal conditions. In some embodiments, the rehydration solution may be composed of a multivalent cation cofactor for catalyzing enzymes and reagents to maintain an optimal pH buffer for an efficient amplification reaction. In one embodiment, the rehydration solution comprises an enzyme catalyzing cofactor, such as Mg+2, Mn+2, or other multivalent cations, which is provided as a cation compound (such as magnesium chloride (MgCh), manganese acetate (Mh(OA ), or any other cation compound) in the rehydration solution at a concentration that is suitable for efficient amplification.
[0186] In some embodiments, a method is provided for detecting a target RNA in a sample, the method comprising:
(a) adding the sample to an amplification mixture comprising:
(i) an RNA replicase (RNA-dependent RNA polymerase);
(ii) at least one target oligonucleotide primer specific for the target RNA;
(iii) nucleoside triphosphates (NTPs) ATP, CTP, GTP and UTP;
(iv) at least one or more of a detectable, target probe for detection of the target RNA; and
(v) optionally, an RNAse inhibitor, one or more of a reaction helper protein, or combinations thereof;
(b) producing at least one or more of an RNA amplicon using RNA replicase-dependent amplification (RDA) of the target RNA, if present in the sample; and
(c) hybridizing one or more target probe(s) to at least one or more of the RNA amplicon to detect presence or absence of the target RNA.
[0187] In various embodiments, the methods disclosed herein relates to direct and/or isothermal amplification of RNA. In some embodiments, the methods comprises direct amplification of RNA, i.e., RNA replicase-dependent amplification (RDA), without formation of a cDNA or DNA. In some embodiments, the methods comprises direct amplification of RNA that is performed essentially isothermally. In some embodiments, the method utilizes an RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) that comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto. In some embodiments, the method utilizes a recombinant RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV). In some embodiments, the recombinant RNA replicase comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto. In various embodiments, a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1, is disclosed herein. A nucleic acid encoding the amino acid sequence of SEQ ID NO: 3, is also disclosed herein. Exemplary nucleic acid sequences of the RNA replicase of the various embodiments disclosed herein can include the nucleic acid sequence set forth in SEQ ID NO: 2, or a nucleic acid sequence having at least 80%, 90%, or 95% sequence identity thereto, and SEQ ID NO: 4, or a nucleic acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
[0188] The components of the amplification mixtures, amplification compositions, compositions and the like, employed in the methods for direct amplification and detection of a target RNA, are similar to those described above. It is understood that, the compositions employed in the methods disclosed herein can further include any reagent that is conventionally employed for nucleic acid amplification, more specifically, for direct and/or isothermal RNA amplification utilizing RDA, including those that are not explicitly disclosed herein.
III. Kris
[0189] In various embodiments, a kit is provided, wherein the kit essentially comprises the compositions described above. In various embodiment, the kit comprises an RNA replicase (RNA- dependent RNA polymerase), at least one target oligonucleotide primer specific for a target RNA to be amplified, nucleoside triphosphates (NTPs) (i.e., ATP, CTP, GTP and UTP), one or more detectable, target probe(s) for detection of the target RNA, and optionally, an RNAse inhibitor, one or more reaction helper protein, or combinations thereof. In some embodiments, the reaction helper protein is a protein that assists in the activity of the RNA replicase (RNA-dependent RNA polymerase), stabilizes the target RNA, or separates a double-stranded RNA into single-stranded RNA. In some embodiments, the reaction helper protein can be an RNA-binding protein (RBP), which
includes, but is not limited to, an RNA chaperone, an RNA helicase, an RNA binding-factor, a scaffold protein, or an RNA-modifying enzyme that may be required for, but not limited to, an RNA replicase activity, target RNA-binding, and/or assisting in stability of a target RNA. In some embodiments, the RNA helicase can be used to separate strands in a duplex (i.e., double-stranded RNA), allowing primers to bind the duplex and be extended during the amplification process.
[0190] In various embodiments, the compositions of the kit, when combined with a sample
(e.g., a test sample) is capable of producing a single-stranded RNA amplicon (ssRNA amplicon), a double-stranded RNA amplicon (dsRNA amplicon), or combinations thereof, using an isothermal RNA replicase-dependent amplification (RDA) of the target RNA, if the target RNA is present in the sample. In some embodiments, the compositions of the kit allow for a direct amplification i.e., RNA replicase-dependent amplification (RDA), of a target RNA without formation of a cDNA or DNA. In some embodiments, the compositions of the kit allow for direct amplification of a target RNA that is performed essentially isothermally. In some embodiments, the compositions of the kit includes an RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) that comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto. In some embodiments, the compositions of the kit includes a recombinant RNA replicase that is derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV). In some embodiments, the compositions of the kit includes a recombinant RNA replicase that comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto. In various embodiments, a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1, is disclosed herein. A nucleic acid encoding the amino acid sequence of SEQ ID NO: 3, is also disclosed herein. Exemplary nucleic acid sequences of the RNA replicase of the various embodiments disclosed herein can include the nucleic acid sequence set forth in SEQ ID NO: 2, or a nucleic acid sequence having at least 80%, 90%, or 95% sequence identity thereto, and SEQ ID NO: 4, or a nucleic acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
[0191] In some embodiments, the kit comprises a first container containing the aforesaid composition, and a second container containing a master mix. In some embodiments, the kit comprises a third container containing a process buffer. In some embodiments, the kit comprises a fourth container containing reagents related to process control. In some embodiments, the compositions of the kit are provided in lyophilized form, frozen liquid form, or combinations thereof. In some embodiments, the kit comprises a fifth container containing a rehydration solution. The kit may also contain instructions for using the aforesaid components and compositions in carrying out the amplification and detection assays disclosed herein.
[0192] Also provided are kits in a single container, containing components of an exemplary reagent composition as described above, e.g., an RNA replicase (RNA-dependent RNA polymerase), at least one target oligonucleotide primer specific for a target RNA to be amplified, nucleoside triphosphates (NTPs) (i.e., ATP, CTP, GTP and UTP), a detectable, target probe for detection of the target RNA, optionally, an RNAse inhibitor, a reaction helper protein, or combinations thereof, and, in some embodiments, one or more stabilizers. In some embodiments, the components of an exemplary reagent composition can be provided as a solid composition in a first container in the kit. [0193] In some embodiments, a container within the kit contains a rehydration solution, for use in rehydrating the solid composition. In some embodiments, the rehydration solution may be composed of a multivalent cation cofactor for catalyzing enzymes. In one embodiment, the rehydration solution comprises an enzyme catalyzing cofactor, such as Mg+2, Mn+2, or other multivalent cations, which is provided as a cation compound (such as magnesium chloride (MgCh). manganese acetate (Mh(OA ), or any other cation compound) in the rehydration solution at a concentration that is suitable for efficient amplification. In one embodiment, the cation compound is provided at a concentration in the rehydration solution of between about 0.1-20 mM, 0.1-10 mM, 0.5- 10 mM, 0.5-8 mM, 0.5-6 mM, 0.1-6 mM, 0.1-5 mM, 0.5-5 mM, 0.1-3 mM, or 0.5-3 mM. In another embodiment, the concentration of manganese acetate in the final assay is between about 0.1-20 mM, 0.1-10 mM, 0.5-10 mM, 0.5-8 mM, 0.5-6 mM, 0.1-6 mM, 0.1-5 mM, 0.5-5 mM, 0.1-3 mM, or 0.5-3 mM. In some embodiments, the rehydration solution may also include reagents to maintain an optimal pH buffer for an efficient amplification reaction or optimal environment for any other proteins and enzymes involved in the methods described herein.
[0194] In some embodiments, the kit may contain a container containing a solution of the process control. External process controls for the pathogens being assayed may also be included. In some embodiments, the kit may comprise a sample processing kit and a master mix container. The sample processing kit may comprise one or more containers comprising process buffer(s) for processing the sample. The master mix kit preferably comprises a container comprising a solid reagent composition, and another container comprising a rehydration solution, for use in rehydrating the solid reagent composition. Other components as described above may also be included such as, but not limited to, a container containing a process control solution and instructions.
[0195] In some embodiments, the solid reagent composition may correspond to any of the selected embodiments described above. The kit may also contain additional containers containing one or more process buffers. In some embodiments, the kit may include a container containing a first process buffer and another container containing a second process buffer. In some embodiments, the first process buffer comprises a sodium azide solution, NaOH, and lithium dodecyl sulfate. In another embodiment, the second process buffer comprises a sodium azide solution, NaCl, Tris, EDTA, a
control sequence, and water qs. In one specific embodiment, the sample processing kit comprises a first and a second processing buffer, a solid reagent composition, and a rehydrating solution, each provided in a separate container.
[0196] In various embodiments, the kit includes software-driven assay protocols for use in commercial nucleic acid amplification instrumentations, when one is employed, which protocols may be provided on a CD. The kit may also contain instructions for using the aforesaid components and compositions in carrying out the amplification and detection assays disclosed herein.
IV. Fusion Proteins and Other Modifications
[0197] In various embodiments, the present disclosure provides a wide variety of engineered and/or recombinant RNA replicase (RNA-dependent RNA polymerase) polypeptides, in particular, an RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV), where the engineered or recombinant molecules optionally comprise a wide array of tags, labels, markers and other types of fusion, modification moieties, and/or modifications such as deletions and additions of domains and segments. It is not intended that the disclosed RNA replicase be limited by the use of any particular tags, labels, or fusion moieties recited herein, as one of skill recognizes that the use of these types of moieties and/or modifications is routine and well within the skill of the ordinary artisan, and further, one of skill will recognize and utilize still other reagents not specifically recited herein, and sill within the spirit of the disclosure.
[0198] In some embodiments, the RNA replicases described herein are fusion polypeptides.
For example, the fusion polypeptides, i.e., of the RNA replicases described herein, can comprise a signal sequence, a detectable moiety, an enzymatic detectable moiety, a detectable label (for example), a detectable particle, a fluorescent label, or any kind of polypeptide tag (for example, a purification tag, such as a poly-HIS tag). In one embodiment, the RNA replicases described herein comprise a poly-histidine tag, in particular, a hexa-histidine peptide (HIS) tag, which provides for convenient purification of the fusion protein. Other polypeptide tags useful for purification include, but are not limited to, the hemagglutanin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein, GST, c-myc and the "flag" tag. Preferably, the appending of a second amino acid sequence as a marker sequence or polypeptide tag to construct the fusion polypeptide does not interfere with the specific activity, e.g., RNA amplification activity, of the modified RNA replicase. Polynucleotides and amino acid sequences encoding these fusion proteins are also encompassed by the disclosure.
[0199] Accordingly, in some embodiments, the disclosure provides RNA replicases having appended sequences to facilitate protein purification. For example, the RNA replicases can be appended with coding sequence for a hexa-histidine peptide (HIS) tag, which allows for convenient purification of the fusion polypeptide, for example, using a Ni-NTA (nitrilotriacetic acid) resin, as
known in the art. See, for example, the RNA replicases of the present disclosure that are appended with HIS tag sequence (see, for example, FIG. 3 and SEQ ID NO: 3). Any of the RNA replicases described herein can be similarly appended.
[0200] In other embodiments, the RNA replicase polypeptides can further comprise operably fused amino acid sequences that encode any number and any type of beneficial functionalities, for example, any type of fused polypeptide marker or tag of any desired sequence, or fused peptide sequences that mark the fusion protein for a particular post-translational modification. The disclosure provides numerous examples of protein markers and protein tags, any of which find use with the compositions and methods described herein. The compositions and methods described herein are not to be limited to polypeptide markers or tags recited herein, as one of ordinary skill recognizes the diversity of markers and tags known in the art that readily find use with the RNA replicase polypeptides described herein.
[0201] In some embodiments, the RNA replicases having appended sequences that program the RNA replicase molecule for extracellular transport, so that the RNA replicase is secreted to the cell culture medium from the host cell. A signal sequence specific for prokaryotic export or eukaryotic export can alternatively be used depending on the application. Any of a variety of signal sequences are available and can be used, as commonly recognized by one of skill in the art. It is not intended that the RNA replicase polypeptides be limited to the particular signal sequences described herein. [0202] It may be necessary to engineer an RNA replicase with a deletion of a domain that is present in the parent polypeptide form from which it was derived to achieve improved solubility during protein purification. In some embodiments, the RNA replicase can be designed with a deletion of the transmembrane domain that is present in the parent polypeptide form (e.g., an RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV)) from which it was derived to achieve improved solubility during protein purification, without interfering with the RNA amplification activity of the modified RNA replicase. See, for example, the RNA replicases of the present disclosure that are designed with a deletion of the transmembrane domain for improved solubility (see, for example, FIG. 3 and SEQ ID NO: 3). Any of the RNA replicases described herein can be similarly designed.
[0203] In other aspects, any combination, any type and any quantity of appended (i.e., fused) amino acid sequences can be used to produce the RNA replicase polypeptides as described herein. For example, these recombinant RNA replicase polypeptides can include one or any number of operably fused polypeptide tags, for example, the HIS-tag for ease of detection and purification, one or any number of fused marker protein, for example, a red fluorescent protein, green fluorescent protein, blue fluorescent protein, or any other kind of fluorescent protein marker. Other types of beneficial fused sequences that encode polypeptides, or even singe amino acids, can also be operably linked to the
RNA replicase polypeptides to produce proteins with additional functionalities. For example, these include but are not limited to, signal sequences for post-translational modifications that may be beneficial for the use of the RNA replicase for purposes of the methods described herein, and unnatural amino acids that have beneficial functionalities such as resistance to degradation (improved half-life of the fusion protein) and chemically reactive side chains.
[0204] In some embodiments, derivatives or fusions of the RNA replicase molecules provided herein are polypeptides that have been altered so as to exhibit additional features or activities not found on the parent polypeptide form (e.g., an RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV)) from which it was derived. Examples include fusion proteins, for example, fusions resulting the RNA replicase molecule also carrying an enzymatic activity to allow detection, for example, a luciferase activity or an alkaline phosphatase activity. The precise site at which the fusion is made to produce the fusion polypeptide or protein may be selected empirically to optimize the secretion or binding characteristics of the fusion protein. DNA encoding the fusion protein is then transfected into a host cell for expression.
V. Nucleic Acid Sequences. Vectors and Host Cells
[0205] In some embodiments, the present disclosure provides, nucleic acid sequences, including recombinant nucleic acid sequences, encoding an RNA replicase (RNA-dependent RNA polymerase), in particular, an RNA replicase derived from the nonstructural protein 5B (NS5B) of hepatitis C virus (HCV), as described herein. It is understood that terms nucleic acid and polynucleotide are used interchangeably herein. These nucleic acid sequences and polynucleotides can be contained on any suitable vector. In some embodiments, the nucleic acid sequence and polynucleotide is operably coupled to at least one regulatory polynucleotide capable of inducing the expression of the RNA replicase in a host cell. The host cells comprising the polynucleotide are also a feature of the compositions and methods of the disclosure.
[0206] In some embodiments, the present disclosure provides polynucleotide sequences encoding the RNA replicase polypeptides described herein, and variants of these sequences. Polynucleotide sequences encoding the RNA replicases described in the present disclosure are provided in FIGS. 2 and 4 and SEQ ID NOS: 2 and 4. It is not intended that polypeptides finding use with the methods described herein be limited to the polynucleotides provided in the present disclosure, as one of skill will recognize that many polynucleotides can be generated that encode any one particular polypeptide, and it is well within the skill of one with ordinary skill to generate alternative polynucleotide sequences encoding the RNA replicases described herein.
[0207] A polynucleotide encoding the RNA replicases described herein can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide encoding an RNA replicase described herein
can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, a polynucleotide encoding an RNA replicase described herein can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide encoding an RNA replicase described herein may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.
[0208] The polynucleotides may be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of the RNA replicase is known, a polynucleotide encoding the RNA replicase may be assembled from chemically synthesized oligonucleotide. Alternatively, a polynucleotide encoding an RNA replicase may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a desired RNA replicase is not available, but the sequence of the RNA replicase molecule is known, a nucleic acid encoding the RNA replicase may be chemically synthesized or obtained from a suitable source by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the RNA replicase. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any suitable method.
Vectors
[0209] In some embodiments, the present disclosure provides vector compositions which contain polynucleotide sequences described herein. Polynucleotides described herein can be incorporated into any desired DNA or RNA based vector, without limitation. For example, a polynucleotide encoding an RNA replicase, can be cloned into a suitable expression vector, a subcloning vector, a shuttle vector, a vector designed for use with in vitro transcription reactions, cosmids, phagemids, and vectors derived from mammalian viruses, including retroviruses (for example, lentiviruses), adenoviruses, and adeno-associated viruses (AAV).
[0210] In some applications, vectors can be conveniently supplied and used in their circular form, or in a linearized form. The linearized form finds use in subcloning steps such as in cloning the guide RNA target sequence into the host vector. Linearized vectors are also used in in vitro transcription reactions.
Expression Vectors
[0211] In some aspects, the disclosure provides a wide variety of expression vectors for expression of various polypeptide products. An expression vector can be optimized to express an
mRNA in any suitable cell type, for example, bacterial cells, mammalian cells, human cells or mouse cells. An expression vector as used herein can be optimally designed to express a protein, e.g., an RNA replicase, in a mammalian host cell. In that embodiment, a vector comprising protein-coding open reading frame and suitable regulatory elements can be delivered into the host cell by any suitable method of transfection or transduction. Within the cell, that ORF is transcribed by endogenous RNA polymerases (RNA pol II in the case where a protein coding gene is expressed) to produce mRNA, and that in turn is translated to produce the encoded protein.
[0212] The term "vector" or "expression vector" is used herein to mean vectors used as a vehicle for introducing into and expressing a desired gene in a host cell. As known to those skilled in the art, such vectors are readily available, for example, selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors can comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells. For the purposes of the compositions and methods described herein, numerous expression vector systems may be employed. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals.
[0213] More generally, once the vector or DNA sequence encoding the RNA replicase has been prepared, the expression vector may be introduced into an appropriate host cell. Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection including lipotransfection using, e.g., Fugene or lipofectamine, protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. Typically, plasmid introduction into the host is via standard calcium phosphate co-precipitation method. The host cells harboring the expression construct are grown under conditions appropriate to the production of the RNA replicase. Promoters and Regulatory Elements
[0214] Polynucleotides of the present disclosure are most typically in the context of a vector, such as an expression vector. The expression vector can be associated with any type and any quantity
of regulatory elements. Such elements can promote the strong and accurate transcription of the polynucleotide, for example, to generate mRNA, either in a cell, or in an in vitro transcription reaction. Regulatory elements can refer to DNA elements that regulate transcription or regulate translation. Regulatory elements can reside upstream of an open reading frame, or downstream of an open reading frame. It is not intended that the compositions and methods described herein be limited to any particular type or sequence of regulatory elements.
[0215] In various aspects, the polynucleotides of the present disclosure, e.g., polynucleotides that encode an RNA replicase, are transcribed within mammalian cells to produce mRNA. When mRNA is to be produced, transcription is directed by an RNA type II polymerase (RNA pol II), which requires a mammalian pol II promoter sequence. The pol II promoter that is used is not limited in any respect. Promoters that function constitutively and promoters that are regulated by induction or repression all find use with the compositions and methods described herein. It is not intended that the compositions and methods described herein be limited to any of the promoters disclosed herein, as one of ordinary skill in the art recognizes that a wide variety of promoters find are available, for example, constitutively active promoters, inducible promoters, repressible promoters, tissue specific promoters and cell-type specific promoters.
[0216] RNA pol II promoters that can drive high levels of protein expression in mammalian cells may be preferred, and most preferably, are active in a wide range of cell types and species. Constitutively active promoters commonly used in mammalian systems include the simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), human ubiquitin C promoter (UBC), human elongation factor 1 alpha promoter and hybrid promoter (EFl-a), mouse phosphogly cerate kinase 1 promoter (PGK), and chicken b-actin promoter coupled with CMV early enhancer (CAG) promoter and murine stem cell virus (MSCV) promoter.
[0217] In some embodiments, it is desirable to the polypeptide of interest only in certain cell types. In those embodiments, the use of tissue specific or cell type specific mammalian promoters can be used, which are knows in the art.
[0218] An expression vector of encompassed by the present disclosure can include translation regulatory elements that control accurate translation of the mRNA to produce protein. For example, a polypeptide ORF can be linked to translation initiation sequences, translation termination sequences and/or other post-transcriptional regulatory sequences, such as the woodchuck virus post- transcriptional regulatory element (WPRE) to boost gene expression and stabilize the mRNA transcript Host Cells
[0219] In some aspects, vectors are located within host cells, and host cells are used to express the polypeptides of interest. Host cells comprising one or more polynucleotide or vector of disclosure
is also provided by the disclosure. The polynucleotide that is contained in the host cell can be of any type, for example, an expression vector or a mRNA molecule that was produced by in vitro transcription, or an mRNA molecule that was produced by in vivo transcription within the host cell using endogenous RNA polymerases. The type of host cells finding use with the compositions and methods described herein are not limited, as many types of host cells can be used.
[0220] In some aspects, bacterial host cells find use, for example, in propagating and producing plasmid DNA, or producing desired polypeptide. Mammalian host cells such as mouse or human cells also find use for producing polypeptides described herein. It is not intended that the disclosure be limited to the any particular host cells described herein, as the disclosure is widely applicable to cells derived from many species, including all primates, mouse, rat, and any other mammalian species, as well as bacterial host systems. Stable mammalian cell lines created using the host cells of the disclosure find use for producing polypeptides of interest, namely, the RNA replicases described herein.
[0221] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an RNA replicase for use in the methods described herein. Thus, the compositions and methods described herein includes host cells comprising a polynucleotide encoding an RNA replicase, which preferably are operably linked to a heterologous promoter.
[0222] As used herein, "host cells" refers to cells which harbor vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions of processes for isolation of recombinant proteins, e.g., engineered and recombinant RNA replicases, from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to denote the source of RNA replicase unless it is clearly specified otherwise. In other words, recovery of polypeptide from the "cells" may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
[0223] The host cell line used for protein expression is often of mammalian origin; those skilled in the art are credited with ability to preferentially determine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/0 (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-lclBPT (bovine endothelial cells), RAH (human lymphocyte) and 293 (human kidney). CHO and 293 cells are particularly preferred. Host
cell lines are typically available from commercial services, the American Tissue Culture Collection or from published literature.
[0224] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells, which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the RNA replicase molecule may be engineered.
[0225] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the RNA replicase molecule being expressed. For example, when a large quantity of such a protein is to be produced, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
VI. Expression and Purification of Recombinant Proteins
[0226] The present disclosure provides compositions and methods for the expression and purification of the RNA replicases described therein. RNA replicases of the present disclosure can be produced by any method known in the art for the synthesis of engineered and recombinant protein molecules, in particular, by chemical synthesis or preferably by recombinant expression techniques as described herein.
[0227] For example, once the appropriate genetic material is obtained, the polynucleotide encoding the RNA replicase can be inserted into expression systems contained on vectors that can be transfected into standard recombinant host cells. A variety of such host cells may be used; for efficient processing, however, mammalian cells are preferred. Typical mammalian cell lines useful for this purpose include, but are not limited to, CHO cells, HEK 293 cells, or NSO cells.
[0228] The production of the RNA replicase is then undertaken by culturing the modified recombinant host under culture conditions appropriate for the growth of the host cells and the expression of the coding sequences. The RNA replicase is then recovered by isolating them from the culture. The expression systems can be designed to include signal peptides so that the resulting recombinant protein are secreted into the medium; however, intracellular production is also possible. [0229] Following manipulation of the isolated genetic material to provide the RNA replicases, the polynucleotides encoding the RNA replicases are typically inserted in an expression vector for
introduction into host cells that may be used to produce the desired quantity of the RNA replicases. Recombinant expression of an RNA replicase is described herein. Once a polynucleotide encoding an RNA replicase molecule of the disclosure has been obtained, the vector for the production of the RNA replicase molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an RNA replicase encoding nucleotide sequence are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors containing an RNA replicase coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The disclosure provides replication-competent vectors comprising a nucleotide sequence encoding an RNA replicase, operably linked to a promoter.
Claims
1. A method for detecting a target RNA in a sample, comprising:
(a) adding the sample to an amplification mixture comprising:
(i) an RNA replicase (RNA-dependent RNA polymerase);
(ii) at least one target oligonucleotide primer specific for the target RNA;
(iii) nucleoside triphosphates (NTPs) ATP, CTP, GTP and UTP;
(iv) at least one or more of a detectable, target probe for detection of the target RNA; and
(v) optionally, an RNAse inhibitor, one or more of a reaction helper protein, or combinations thereof;
(b) producing at least one or more of an RNA amplicon using RNA replicase-dependent amplification (RDA) of the target RNA, if present in the sample; and
(c) hybridizing the at least one or more of the detectable, target probe to the at least one or more of the RNA amplicon to detect presence or absence of the target RNA.
2. The method of claim 1, wherein the amplification mixture further comprises a control oligonucleotide primer pair specific to a process control sequence and a control probe for detection of the process control sequence.
3. The method of claim 2, wherein the at least one target oligonucleotide primer and control oligonucleotide primer pair are designed such that amplification and detection of the target RNA and the process control sequence can be performed simultaneously using the same amplification conditions.
4. The method of any preceding claim, wherein the at least one or more of the target probe is a single-strand probe that hybridizes to the ssRNA amplicon, one strand of the dsRNA amplicon, or both strands of the dsRNA amplicon.
5. The method of claim 4, wherein the at least one or more of the target probe is an optically- labeled probe.
6. The method of claim 5, wherein the optically-labeled probe comprises a fluorescent compound.
7. The method of claim 6, wherein the amplification is real-time amplification and producing the at least one or more of the RNA amplicon is determined real-time.
8. The method of claim 7, wherein producing the at least one or more of the RNA amplicon is
determined by an end-point analysis.
9. The method of claim 8, wherein the end-point analysis is achieved by a lateral-flow device.
10. The method of any preceding claim, wherein said RDA is performed essentially isothermally.
11. The method of any one of claims 1-10, wherein the amplification mixture comprises a single target oligonucleotide primer.
12. The method of any one of claims 1-10, wherein the amplification mixture comprises a first target oligonucleotide primer and a second target oligonucleotide primer.
13. The method of claim 12, wherein the target RNA is a double-stranded RNA, and wherein the first and second target oligonucleotide primers hybridize to opposite strands of the double-stranded target RNA in 5’ to 3’ orientation.
14. The method of any preceding claim, wherein the RNA replicase is a recombinant RNA replicase (RNA-dependent RNA polymerase).
15. The method of claim 14, wherein the recombinant RNA replicase (RNA-dependent RNA polymerase) is derived from nonstructural protein 5B (NS5B) of hepatitis C virus (HCV).
16. The method of claim 15, wherein the recombinant RNA replicase (RNA-dependent RNA polymerase) comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
17. The method of any preceding claim, wherein presence of the at least one or more of the RNA amplicons is diagnostic for a pathogen or a mutation in a genomic RNA.
18. A composition for detecting a target RNA in a sample, comprising:
(i) an RNA replicase (RNA-dependent RNA polymerase);
(ii) at least one oligonucleotide primer specific for the target RNA;
(iii) nucleoside triphosphates (NTPs) ATP, CTP, GTP and UTP;
(iv) at least one or more of a detectable, single-strand probe that hybridizes to the target RNA and an RNA amplicon thereof; and
(v) optionally, an RNAse inhibitor, one or more of a reaction helper protein, or combinations thereof; wherein said composition when combined with a sample is capable of producing a single- stranded RNA amplicon (ssRNA amplicon), a double-stranded RNA amplicon (dsRNA amplicon), or combinations thereof, using an isothermal RNA replicase-dependent amplification (RDA) of the target RNA, if present in the sample.
19. The composition of claim 18, wherein the RNA replicase is a recombinant RNA replicase
derived from nonstructural protein 5B (NS5B) of hepatitis C virus (HCV).
20. The method of claim 19, wherein the recombinant RNA replicase comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
21. A recombinant RNA replicase (RNA-dependent RNA polymerase) comprising the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 80%, 90%, or 95% sequence identity thereto.
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