WO2021080629A1 - Procédés d'amplification par déplacement de brin isotherme véritable - Google Patents

Procédés d'amplification par déplacement de brin isotherme véritable Download PDF

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WO2021080629A1
WO2021080629A1 PCT/US2019/065210 US2019065210W WO2021080629A1 WO 2021080629 A1 WO2021080629 A1 WO 2021080629A1 US 2019065210 W US2019065210 W US 2019065210W WO 2021080629 A1 WO2021080629 A1 WO 2021080629A1
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nucleic acid
sequence
target nucleic
amplification
primer
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PCT/US2019/065210
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English (en)
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Yevgeniy S. Belousov
Eugeny A. Lukhtanov
Noah Scarr
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Elitechgroup, Inc.
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Priority claimed from US16/660,961 external-priority patent/US10975423B2/en
Application filed by Elitechgroup, Inc. filed Critical Elitechgroup, Inc.
Publication of WO2021080629A1 publication Critical patent/WO2021080629A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

  • This disclosure pertains to methods for isothermal strand displacement amplification that accomplishes efficient primer extension amplification with target specific primers and does not require pre-denaturation.
  • Isothermal amplification requires single stranded targets for efficient primer extension.
  • Helicase dependent amplification of nucleic acids also requires helicase enzyme for unwinding double strands to allow amplification with a DNA polymerase (US Patent No. 7,282,328).
  • Exponential strand displacement amplification (“SDA”) as described in US Patent No. 5,455,166 requires an initial denaturation of the target into single-stranded DNA (ssDNA), generation of hemiphosphorothioate sites which allow single strand nicking by restriction enzymes, and extension by a polymerase lacking 5’ -3’ exonuclease activity.
  • Raising the temperature of the reaction to approximately 95°C to render double strands into single strands is required to permit binding of the primers to the target strands.
  • State of the art SDA amplification requires the denaturation of the target at elevated temperature to yield ssDNA for strand displacement isothermal amplification.
  • ds double stranded nucleic acid
  • Heat denaturation is state of the art to separate ds DNA into single strands.
  • Native DNA denatures at about 85°C. (White, Handler and Smith, Principles of Biochemistry 5 th Edition, McGraw-Hill Kogakush, Ltd, pages 192-197, 1993).
  • primer extension in amplification required the binding of a primer to a single strand DNA strand. This was preferably achieved by heating the sample at about 95°C (M Panaccio and A Lew. PCR based diagnosis in the presence of 8% (v/v) blood. Nucleic Acids Res., 19: 1151 (1991)). It was recently reported that Watson-Crick pairs in naked DNA spontaneously flip into Hoogstein pairs under ordinary conditions, suggesting that DNA breathes (Fran- Kamentskii. Artificial DNA; PNA & XNA, 2:1, 1-3 (2011)).
  • the present invention relates generally to an isothermal assay which utilizes the advantages of target nucleic acid amplification without the requirement for dsDNA denaturation.
  • the present methods enable efficient detection of target nucleic acids with extraordinar specific amplification.
  • the present disclosure unexpectedly determined that primers designed according to a particular method allow efficient primer extension amplification of target specific primers without pre-denaturation.
  • the present disclosure provides methods, primers and probes for the isothermal amplification without denaturation of nucleic acid targets for polymerase primer extension (isothermal strand displacement amplification (“iSDA”)) in samples including biological samples (e.g., blood, nasopharyngeal or throat, swab, wound swab, or other tissues).
  • the nucleic acid targets may be double stranded or they may be single stranded, such as RSV virus.
  • RNA targets may be single stranded or double stranded.
  • the method described herein utilizes primer oligonucleotides that allow primer extension without denaturation of nucleic acid targets.
  • the primers have modified bases to improve stability or to eliminate primer self-association.
  • modified bases are used to limit primer self-association.
  • the primer comprises a 5’-non-complementary tail wherein said tail further comprises a nicking enzyme specific sequence.
  • the nucleic acids present in a clinical or test sample obtained from a biological sample or tissue suspected of containing a clinical target are extracted with methods known in the art.
  • the target nucleic acids are amplified without denaturation and detected. More specifically the target specific primers contain a sequence specific for target and a non-target complementary 5’ -tail, wherein the tail contains a sequence specific for a nicking enzyme when hybridized to its complementary sequence. At least one amplification cycle provides a double stranded amplicon containing a nicking site which allows strand displacement in a second amplification cycle.
  • the amplified nucleic acid can be detected by a variety of state of the art methods including fluorescence resonance energy (“FRET’), radiolabels, lateral flow, enzyme labels, and the like.
  • FRET fluorescence resonance energy
  • the methods described herein also include methods for the design of primers allowing amplification of at least one cycle of amplification without denaturation of duplex DNA target.
  • the methods comprise the detection of iSDA or RT-iSDA amplified targets by lateral flow.
  • This disclosure provides an isothermal method for specifically detecting a nucleic acid sequence in a biological sample from an individual.
  • the disclosure also provides oligonucleotide primers and probes comprising nucleotide sequences characteristic of specific genomic nucleic acid sequences.
  • the method includes performing isothermal amplification without a denaturation step prior to amplification.
  • the amplification step includes contacting the sample nucleic acid with pairs of primers to produce amplification produces) if the specific genomic nucleic acid target is present
  • the preferred primers target a specific region of a specific target gene.
  • Each of the preferred primers has a 5’ -oligonucleotide tail noncomplementary to the target where said non-complementary tail contains a sequence when hybridized to a complementary sequence contains a nicking enzyme cleavage site.
  • the oligonucleotide probes detect the amplified target directly or indirectly.
  • the preferred oligonucleotide probe is a S’-minor groove binder-fluorophore-oligonucleotide-quencher-3’ conjugate that fluoresces on hybridization to its complementary amplified target.
  • one or more primer is labeled.
  • a double strand binding fluorescent dye is used.
  • one or more bumper oligonucleotides are provided.
  • the probe(s) is omitted.
  • the amplified target is captured on a solid support or membrane and detected by a labeled probe.
  • the primer concentrations are present in different concentrations.
  • an internal control is provided.
  • Figure 1 shows a schematic of an example of dual capture and detection of iSDA amplified amplicon by pDNA immobilized on a solid surface.
  • Figure 2 shows an example of real-time iSDA amplification of different concentrations of the Idhl gene with fluorescence detection utilizing a Pleiades probe.
  • Figure 3 shows an example of lateral flow colorimetric detection of an Idhl iSDA amplified amplicon with the approach provided in Figure 1.
  • Figure 4 shows an example of real-time iSDA amplification of two different mecA designed assays with fluorescence detection utilizing a Pleiades probe.
  • Figure 5 shows an example of real-time iSDA amplification with different polymerases.
  • Figure 6 shows an example of gel analysis of the valuation of Nt.Alw I on PCR Amplified target containing NtAlw I cleavage site.
  • Figure 7 shows an example of lateral flow detection of iSDA biplex- amplified Idhl and IC amplicons.
  • Figure 8 shows a schematic representation of a primer containing a complementary- and non-complementary-sequence.
  • Figure 9 shows the probe specific iSDA detection and differentiation of Idhl gene in S. aureus and of S. epidermis.
  • Figure 10 shows the specific real-time iSDA amplification of S. aureus nucleic acid extracted with five different extraction methods.
  • Figure 11 shows the results of amplification reactions comparing amplification with primers and probes optimized for use in the present isothermal strand displacement amplification method and traditional primers and probes.
  • Figure 12 shows the specific reverse transcriptase-iSDA (RT-iSDA) amplification of Respiratory syncytial vims (RSV) extracted RNA nucleic acid using both realtime fluorescence detection and post-amplification lateral flow detection.
  • RT-iSDA reverse transcriptase-iSDA
  • Figure 13 shows the real-time iSDA amplification of native and denatured Plasmodium falciparum DNA.
  • Figure 14 shows estimated fractions of dissociated bases within subregions of Influenza A vims segment 7 matrix protein 2 (M2) and matrix protein 1 (Ml) genes with varying salt and temperature.
  • Figure 15 shows estimated fractions of dissociated bases within a target mecA sequence and placement of primers designed for iSDA amplification.
  • Figure 16 shows images of chips obtained from digital FCR in iSDA of Idhl target gene using different target concentrations.
  • Figure 17 shows estimated fractions of dissociated bases within a target CMV sequence and placement of primers designed for iSDA amplification.
  • Figure 18 estimated fractions of dissociated bases within a target mecA sequence and placement of primers designed for iSDA amplification.
  • Figure 19 shows estimated fractions of dissociated bases within a target RSV sequence and placement of primers designed for iSDA amplification.
  • Figure 20 shows estimated fractions of dissociated bases within a target IC2 sequence and placement of primers designed for iSDA amplification.
  • Figure 21 A shows estimated fractions of dissociated bases within a target enterovirus sequence and placement of primers designed for iSDA amplification.
  • Figure 2 IB shows an image of a gel indicating detection results for different primers designed for iSDA amplification of a target enterovirus sequence.
  • Figure 22 shows estimated fractions of dissociated bases within a target influenza A H3N1 sequence and placement of primers designed for iSDA amplification.
  • Figure 23A shows a gel image comparing amplification results from a set of primers in a low dissociation region and primers in a region of higher dissociated bases (L1E1) and a set of primers both in regions with a greater estimated fraction of dissociated bases (L2E1).
  • Figure 23B shows a gel image of titration of influenza A virus subtype H3N1 from 3 to 300 copies/reaction.
  • Figure 23C shows a gel image of a titration of influenza A vims subtype H3N1 at 50 copies/reaction in the presences of 10 to 100 ng of human genomic DNA.
  • Figure 24A shows a plot of estimated fraction of dissociated bases versus temperature for an exemplary subsequence.
  • Figure 24B shows a plot of estimated fraction of dissociated bases versus temperature for an exemplary subsequence.
  • Figure 25A shows an example structure of a typical Endonuclease IV probe (SEQ ID NO:83) and enhancer (SEQ ID NO:84) detection system.
  • Figure 25B shows an exemplary probe containing the abasic spacer as disclosed herein (SEQ ID NO: 82) for use with Endonuclease IV.
  • Figure 26A shows melting temperature of each portion of a digested probe containing the abasic spacer (SEQ ID NO:82) according to preferred embodiments disclosed herein.
  • Figure 26B shows a meltcurve analysis of a typical undigested Endonuclease IV probe (“Endo IV”) compared to an undigested probe containing the abasic spacer (“Ap Probe”) according to preferred embodiments disclosed herein, both with a synthetic complement.
  • Endo IV an undigested Endonuclease IV probe
  • Ap Probe an undigested probe containing the abasic spacer
  • Figure 27 shows estimated fraction dissociated for bases in echovirus sequence and design of primers and probes for iSDA amplification and detection.
  • Figure 28A shows signal strength in iSDA detection using Pleiades probes with interrogation of high concentration echovirus samples and with non-template control
  • Figure 28B shows signal strength in iSDA detection using Pleiades probes with lower target echovirus sample concentration and with NTC.
  • Figure 29 shows signal strength in iSDA detection using an Endonuclease IV probe enhancer detection system at low target echovirus sample concentration and with NTC.
  • Figure 30 shows iSDA detection of echovirus amplified target (about 100 copies/reaction) by a Endonuclease IV probe system (“Endo IV”) compared to a probe including the abasic spacer according to preferred embodiments disclosed herein (“Ap Probe”), with NTC.
  • Endo IV Endonuclease IV probe system
  • Ap Probe abasic spacer according to preferred embodiments disclosed herein
  • Figure 31A shows results of amplification using a Pleiades probe for detection of echovirus at 100 target copies per reaction, with NTC.
  • Figure 3 IB shows results of amplification using a probe containing the abasic spacer according to preferred embodiments described herein for detection of echovirus at 100 target copies per reaction, with NTC.
  • Figure 32 shows a comparison of reactions of a typical Endonuclease IV probe system (“Endo IV”) and a probe containing the abasic spacer according to preferred embodiments (“Ap Probe”) with synthetic template, with NTC.
  • Endo IV Endonuclease IV probe system
  • Ap Probe a probe containing the abasic spacer according to preferred embodiments
  • Figure 33 A shows reaction of a probe containing the abasic spacer, Spacer 1, according to preferred embodiments disclosed herein with a synthetic template, with NTC.
  • Figure 33B shows reaction of a probe containing the abasic spacer, Spacer 2, according to preferred embodiments disclosed herein with a synthetic template, with NTC.
  • Figure 33C shows reaction of a probe containing the abasic spacer, Spacer 3, according to preferred embodiments disclosed herein with a synthetic template, with NTC.
  • Figure 33D shows reaction of a probe containing the abasic spacer, Spacer 4, according to preferred embodiments disclosed herein with a synthetic template, with NTC.
  • Figure 34 shows formation of side products measured on Agarose gel at 10 and 11 minutes for iSDA detection using a probe containing the abasic spacer according to preferred embodiments disclosed herein at different potassium phosphate concentrations.
  • Figure 35 shows a comparison of iSDA detection using probes with abasic spacers according to preferred embodiments disclosed herein (“Ap Probe”) with real-time FCR amplification (“RT-PCR”) of a 10 fold titration of a high copy number echovims sample with four replicates.
  • Ap Probe abasic spacers according to preferred embodiments disclosed herein
  • RT-PCR real-time FCR amplification
  • Figure 36A shows signal generation of iSDA amplification (“iSDA”) using probes with abasic spacers according to preferred embodiments disclosed herein compared to real time PCR amplification (“RT-PCR”) at 25 copies/reaction with four replicates.
  • iSDA iSDA amplification
  • RT-PCR real time PCR amplification
  • Figure 36B shows signal generation of iSDA amplification (“iSDA”) using probes with abasic spacers according to preferred embodiments disclosed herein compared to real time PCR amplification (“RT-PCR”) at 12 copies/reaction with four replicates.
  • iSDA iSDA amplification
  • RT-PCR real time PCR amplification
  • Figure 36C shows signal generation of iSDA amplification (“iSDA”) using probes with abasic spacers according to preferred embodiments disclosed herein compared to real time PCR amplification (“RT-PCR”) at 10 copies/reaction with four replicates.
  • iSDA iSDA amplification
  • RT-PCR real time PCR amplification
  • Figure 36D shows signal generation of iSDA amplification (“iSDA”) using probes with abasic spacers according to preferred embodiments disclosed herein compared to real time PCR amplification (“RT-PCR”) at 25 copies/reaction with four replicates.
  • iSDA iSDA amplification
  • RT-PCR real time PCR amplification
  • Figure 37A shows the structure of the Eclipse Dark Quencher.
  • Figure 37B shows the structure of fluorescein-labeled uridine.
  • the present disclosure provides methods, primers and probes for the isothermal amplification and detection, without denaturation, of double stranded nucleic acid targets for polymerase strand displacement amplification (“iSDA”).
  • iSDA polymerase strand displacement amplification
  • the methods and compositions disclosed are highly specific for nucleic acid targets with high sensitivity, specificity and speed that allow detection of clinical relevant target levels.
  • the methods and compositions can easily be used to amplify or detect nucleic acid targets in biological samples.
  • primers can be designed using the Vienna Folding Package (tbi.univie.ac.at.rivo/RNA/) that identifies analyzes sequences that allowing one to minimize the accumulation of non-predictable byproducts especially for longer incubation times and low concentrations of initial template DNA.
  • Vienna Folding Package is a software product that predicts a secondary structure of the primers based on the calculations of the minimum free energy of the hybridization reaction and calculates the probabilities of alternative DNA/DNA duplex structures.
  • Primers designed using software such as the Vienna Folding Package are considered to have an improved hybridization stringency, and thus permit efficient elongation of a target sequence.
  • the T m of the selected primers can then be adjusted by calculation with a preferred software package, such as the Eclipse Design Software 2.3 (Afonina et al., Single Nucleotide Polymorphism Detection with fluorescent MGB Eclipse Systems in A-Z of Quantitative PCR, Ed. S. A. Bustin, International University Line, La Jolla, CA, pages 718-731 andXn- ⁇ , 2004; see also U.S. Patent Nos. 6683173 and 7751982).
  • the software adjusts the Tm of the primers for optimum extension as well, by calculating duplex stabilities using an algorithm applying a nearest-neighbor model for duplex formation thermodynamics for each of the neighboring base pairs.
  • Each nearest neighbor thermodynamic parameter defines a thermodynamic contribution of two corresponding neighboring bases.
  • a preferred oligonucleotide primer sequence is then selected having the desired duplex stability.
  • the primers can also be designed, if necessary or desired, to include modified bases (see US 7,045,610; US 6,127,121; US 6,660,845; US 5,912,340 and US Application Publication No. 2010/057862, all incorporated by reference).
  • the same software package such as Eclipse Design Software 2.3 can be used.
  • sample refers to a sample of any source which is suspected of containing a target sequence. These samples can be tested by the methods described herein.
  • a sample can be from a laboratory source or from a non-laboratory source.
  • a sample may be suspended or dissolved in liquid materials such as buffers, extractants, solvents, and the like.
  • Samples also include biological samples such as plant, animal and human tissue or fluids such as whole blood, blood fractions, serum, plasma, cerebrospinal fluid, lymph fluids, milk, urine, various external secretions of the respiratory, intestinal, and genitourinary tracts, tears, and saliva; and biological fluids such as cell extracts, cell culture supernatants, fixed tissue specimens, and fixed cell specimens.
  • Samples include nasopharyngeal or throat swabs, stools, wound or rectal swabs.
  • Biological samples may also include sections of tissues such as biopsy and autopsy samples or frozen sections taken for histological purposes.
  • a biological sample is obtained from any animal including, e.g., a human.
  • a biological sample may include human and animal pathogens that includes microbes or microorganisms such as a viruses, bacteria, or fungi that causes disease in humans.
  • Biological samples may further also include products of gene mutated-metabolic disorders.
  • flap primer or “overhang primer” refer to a primer comprising a 5’ sequence segment non-complementary to a target nucleic acid sequence, wherein said tail further comprises a nicking enzyme specific sequence and a 3’ sequence segment complementary to the target nucleic acid sequence
  • the flap primers are suitable for primer extension or amplification of the target nucleic acid sequence
  • the primers may comprise one or more non-complementary or modified nucleotides (e.g., pyrazolopyrimidines as described in US 7,045,610 which is incorporated herein by reference) at any position including, e.g., the 5’ end.
  • iSDA isothermal strand displacement amplification
  • fluorescent generation probe refers either to a) an oligonucleotide having an attached minor groove binder, fluorophore, and quencher, b) an oligonucleotide having an attached fluorophore, and quencher, c) an oligonucleotide having an attached minor groove binder, and fluorophore, d) an oligonucleotide having an attached fluorophore and quencher, e) an oligonucleotide having an attached fluorophore, or f) a DNA binding reagent.
  • the probes may comprise one or more non-complementary or modified nucleotides (e.g., pyrazolopyrimidines as described in US 7,045,610) at any position including, e.g., the 5’ end.
  • the fluorophore is attached to the modified nucleotide.
  • the probe is cleaved to yield a fluorescent signal.
  • modified bases increase thermal stability of the probe-target duplex in comparison with probes comprised of only natural bases (i.e., increase the hybridization melting temperature of the probe duplexed with a target sequence).
  • Modified bases can decrease probe and primer self-association compared to only normal bases.
  • Modified bases include naturally-occurring and synthetic modifications and analogues of the major bases such as, for example, hypoxanthine, 2-aminoadenine, 2-thiouracil, 2-thiothymine, inosine, 5- N 4 -ethenocytosine, 4-aminopyrrazolo[3,4-d]pyrimidine and 6-amino-4-hydroxy-[3,4- d]pyrimidine.
  • Any modified nucleotide or nucleotide analogue compatible with hybridization of probe with a nucleic acid conjugate to a target sequence is useful, even if the modified nucleotide or nucleotide analogue itself does not participate in base-pairing, or has altered basepairing properties compared to naturally-occurring nucleotides.
  • modified bases are disclosed in U.S. Pat. Nos. 7,045,610; 5,824,796; 6,127,121; 5,912,340; and PCT Publications WO 01/38584; WO 01/64958, each of which is hereby incorporated herein by reference in its entirety.
  • Preferred modified bases include 5-hydroxybutynyl uridine for uridine; 4-(4, 6-Diamino- 1 H-pyrazolo [3 ,4-d]pyrimidin-3 -yl)-but-3-yn- 1 -ol, 4-amino- ‘H-pyrazolo[3,4- d]pyrimidine, and 4-amino- 1 H-pyrazolo[3 ,4-d]pyrimidine for adenine; 5-(4-Hydroxy-but-l- ynyl)-lH-pyrimidine-2,4-dione for thymine; and 6-amino- 1 H-pyrazolo[3,4-d]pyrimidin- 4(5H)-one for guanine.
  • modified bases are "Super A®: 4-(4,6-Diamino- lH-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-l-ol," "Super G®: 4-hydroxy-6-amino pyrazolopyrimidine” (www.elitechgroup com) and "Super T®: 5 (4 hydroxy but l ynyl)-lH- pyrimidine-2,4-dione”.
  • Super-DTM 3-Alkynyl pyrazolopyrimidine” analogues as universal bases are disclosed in U.S. Patent Application Publication No. 2012/0244535, incorporated by reference.
  • fluorescent label or “fluorophore” refer to compounds with a fluorescent emission maximum between about 400 and about 900 nm. These compounds include, with their emission maxima in nm in brackets, Cy2TM (506), GFP (Red Shifted) (507), YO-PROTM-!
  • quenchers are described in U.S. Patent No. 6,727,356, incorporated herein by reference.
  • Other quenchers include bis azo quenchers (U.S. Patent No. 6,790,945) and dyes from Biosearch Technologies, Inc. (provided as Black HoleTM Quenchers: BH-1, BH-2 and BH-3 quenchers), Dabcyl, TAMRA and carboxytetramethyl rhodamine.
  • linker refers to a moiety that is used to assemble various portions of the molecule or to covalently attach the molecule (or portions thereof) to a solid support, surface or membrane.
  • a linker or linking group has functional groups that are used to interact with and form covalent bonds with functional groups in the ligands or components (e.g., fluorophores, oligonucleotides, minor groove binders, or quenchers) of the conjugates described and used herein.
  • linking groups are also those portions of the molecule that connect other groups (e.g., phosphoramidite moieties and the like) to the conjugate.
  • a linker can include linear or acyclic portions, cyclic portions, aromatic rings, and combinations thereof.
  • solid support refers to any support that is compatible with oligonucleotide attachment, including, for example, glass, controlled pore glass, polymeric materials, polystyrene, beads, coated glass, and the like.
  • MGB, FL, Q, CPG, and ODN refer to “minor groove binder,” “fluorescent label” or “fluorophore ,” “quencher,” “controlled pore glass” (as an example of a solid support), and “oligonucleotide” moieties or molecules, respectively, and in a manner which is apparent from context.
  • probe and conjuggate are used interchangeably and refer to an oligonucleotide having an attached minor groove binder, fluorophore, and quencher.
  • oligonucleotide refers to a compound comprising nucleic acid, nucleotide, or its polymer in either single- or double-stranded form, e.g., DNA, RNA, analogs of natural nucleotides, and hybrids thereof.
  • the terms encompass polymers containing modified or non- naturally-occurring nucleotides, or to any other type of polymer capable of stable base-pairing to DNA or RNA including, but not limited to, peptide nucleic acids as described in Nielsen et al., Science, 254:1497-1500 (1991), bicyclo DNA oligomers as described in Bolli et al., Nucleic Acids Res., 24:4660-4667 (1996), and related structures. Unless otherwise limited, the terms encompass known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally-occurring nucleotides.
  • a “subsequence” or “segment” refers to a sequence of nucleotides that comprise a part of a longer sequence of nucleotides.
  • nucleotides may include analogs of natural nucleotides which exhibit preferential binding to nucleotides other than naturally occuring DNA or RNA; an example of such nucleotides is pDNA (Eschenmoser et al, Helvetica Chimica Acta, “Why Pentose- and Hexose-Nucleic Acids?”, pp. 76: 2161-2183 (1993)).
  • the term “Nicking Enzyme (or nicking endonuclease)” describes an enzyme that cuts one strand of a double-stranded DNA at a specifically recognition recognized nucleotide sequences known as a nicking site. Such enzymes hydrolyse (cut) only one strand of the DNA duplex, to produce DNA molecules that are “nicked”, rather than cleaved.
  • nicking enzymes include N.Alw I, Nb.BbvCl, NLBbvCl, Nb.BsmI, NLBsmAI, NtBspQI, Nb.BsrDI, NtBstNBI, Nb.BstsCI, NtCviPII, Nb.Bpul0I, NLBpulOI and Nt.Bst9I which are commercially available from www.neb.com, www.fermentas.com and www.sibenzyme.com, respectively.
  • the New England Biolabs REBASE website (rebase.neb.com/cgi- bin/azlist?nick) lists 917 nicking enzymes.
  • “Nicking Enzyme” also includes engineered enzymes that cut one strand of a double stranded DNA for example zinc finger nucleases.
  • the term “Lateral Flow” describes a porous membrane capable of nonabsorbent lateral flow used as assay substrate; a member of the binding pair is affixed in an indicator zone defined in the substrate.
  • the sample is applied at a position distant from the indicator zone and permitted to flow laterally through the zone; any analyte in the sample is complexed by the immobilized specific binding member, and detected.
  • Lateral flow utilizing immuno-binding pairs is well known in the art (US 4,943,522).
  • Lateral flow using DNA binding pairs was disclosed in US US7488578.
  • pDNA binding pairs are disclosed in co-owned US application 2012-0015358 Al.
  • Biotin-streptavidin affinity pairs are well known in the art and commercially available.
  • Streptavidin-coated label may be a covalent or adsorptively bound streptavidin or other biotin-binding species, and the label may be a polystyrene nanoparticle doped with fluorescent or visible dye, a carbon black nanoparticle, a metal colloid, or other species detectable by fluorescence, radioactivity, magnetism, or visual acumen.
  • the lateral flow buffer may be an aqueous suspension containing detergents, proteins, surfactants, and salts.
  • the lateral flow strip may be a porous matrix composed of nitrocellulose, modified nitrocellulose, polyethersulfone, cellulose, glass fiber, polyvinylidene fluoride, or nylon.
  • the lateral flow strip has at least one detection region composed of affinity pairs specific to the iSDA reaction products.
  • this disclosure provides an isothermal method for specifically detecting a nucleic acid sequence in a biological sample from an individual.
  • the isothermal method can be carried out entirely at room temperature, or between about 40°C and about 65°C, or more preferably between about 45°C and about 55°C.
  • the disclosure also provides oligonucleotide primers and probes comprising nucleotide sequences characteristic of a specific genomic nucleic acid sequences.
  • the method includes performing of isothermal amplification without a denaturation step prior to amplification.
  • the amplification step includes contacting the sample nucleic acid with pairs of primers to produce amplification product(s) if the specific genomic nucleic acid target is present.
  • the primer “A-B” comprises a complementary sequence “B” and comprises a non-complementary nicking enzyme recognition sequence site “A” when hybridized to a complementary sequence ( Figure 8).
  • Primer A-B further comprises sequences selected by free energy minimization for specific hybridization and efficient elongation.
  • the primers target a specific region of a specific target gene that allows amplification without thermal denaturation.
  • Bumper primers hybridize upstream of the 5’-end of the flap primers to generate a target specific single stranded DNA newly synthesized amplicon by strand displacement (Nuovo GJ, .Diagn Mol Pathol. 2000 Dec;9(4): 195-202.).
  • the oligonucleotide probes detect the amplified target directly or indirectly.
  • the preferred oligonucleotide probe is a S’-minor groove binder-fluorophore- oligonucleotide-quencher-3’ conjugate that fluoresces on hybridization to its complementary ampl
  • the probe(s) is omitted.
  • the amplified target is captured on a solid support, surface or membrane and detected by a labeled probe.
  • the primer concentrations are present in different concentrations.
  • an internal control is provided.
  • human, animal, and/or plant pathogen nucleic acids are amplified and detected.
  • the amplified target nucleic acid is RNA and the method further comprises a reverse transcriptase step.
  • the S’ non-complementary sequence comprises a sequence for a nicking site.
  • preferred nicking enzyme recognition sequences are selected from N.Alw I, Nb.BbvCl, NLBbvCl, Nb.BsmI, NLBsmAI, NLBspQI, Nb.BsrDI, NLBstNBI, Nb.BstsCI, NtCviPH, Nb.BpulOI, NLBpulOI and Nt.Bst9I, Nb.Mva 12691 and endo nuclease V.
  • a complementary primer sequence comprises a sequence with an Endonuclease V (“Endo V”) cleavage site requiring no heat or chemical denaturation, as more fully described in U.S. Patent No. 8,202,972 or U.S. Patent Application Publication No. 2011/0171649 incorporated by reference, which describes Endo V-based amplification primers. More specifically Endonuclease V is a repair enzyme recognizing DNA oligonucleotides containing deaminated modified bases such as inosine. Endo V cleaves the second or third phosphodiester bond 3’ to the modified base, such as inosine.
  • Endonuclease V is a repair enzyme recognizing DNA oligonucleotides containing deaminated modified bases such as inosine. Endo V cleaves the second or third phosphodiester bond 3’ to the modified base, such as inosine.
  • 8,202,972 describes an Endonuclease V-based amplification method that extends a forward- and reverse-primer containing inosine adjacent to 3’ -end terminal base.
  • the Endo V cleaves the second or third phosphodiester bond 3’ to the inosine in the same strand.
  • the 3’-hydroxyl of the nick is extended by DNA polymerase in a template- directed manner.
  • U.S. Patent No. 8,202,972 requires that “target dsDNA may be thermally denatured, chemically denatured, or both thermally and chemically denatured”.
  • the primers used in the isothermal strand displacement amplification (iSDA) methods are designed to first require the identification of sequences in double-stranded nucleic acids (NA) where Watson-Crick pairs spontaneously flip into Hoogsteen pairs under ordinary conditions, a phenomenon that has led to the suggestion that DNA “breathes” (Fran-Kamentskii (2011)). According to Ehses et al
  • primers can be designed using the Vienna Folding
  • the Vienna Folding Package can be used to predict a secondary structure of NA sequences, including primers, based on the calculations of the minimum free energy of the hybridization reaction and to calculate the probabilities of alternative DNA/DNA duplex structures. Due to the potential interactions amongst primer sequences, some assay designs work significantly better than others. An example of that is seen in FIG. 4, where the mecA design 1 at 50 copies shows a Ct of about 15 while design 2 at the same concentration shows a Ct of 8. Assays for iSDA designed with this software product can show little or no amplification.
  • DNA denaturation and bubble formation in ds nucleic acids can be modelled by various methods.
  • a prominent method uses the Peyrard Bishop Dauxois (PBD) model (Dauxois et al., 1993), based on Langevin equations including the following parameters: Morse potential for hydrogen bonding, adjacent base-pair stacking interactions, thermal noise and other sequence-independent parameters.
  • PBD Peyrard Bishop Dauxois
  • a variation of the PBD model is the helicoidal model which addresses torque-induced denaturation.
  • Another alternative is the Tru- Scheraga free energy model, described by Metzler et al., 2009. These methods employ cooperativity factors for ranges of polymer length to describe local denaturation.
  • Tm prediction parameters which are well-established (SantaLucia, Jr. (1998)) for DNA, can be applied to subsequences in a longer DNA sequence. Specifically, enthalpy and entropy values for nearest neighbors are calculated for each subsequence in an ordered walk to create a profile of interstitial stability along the length of the entire sequence. Short-range (as short as two nucleobases) can be combined with longer (50 nucleobases or more) subsequences to account for long range effects mimicking cooperativity in the PBD and Tru-Scheraga models.
  • Salt conditions can be used to generate a predicted Tm for each subsequence, and growth rate of the dissociation curve can be estimated based on enthalpy values (Mergny and Lacroix, (2003)).
  • enthalpy values Mergny and Lacroix, (2003).
  • the fraction of associated base pairs can be calculated for each subsequence at a particular temperature, and the values plotted over the length of the entire sequence of interest for parameters such as salt content or temperature of analysis as shown in FIG. 14. Sequences with a higher estimated fraction of dissociation allow for the favorable design of primers that can hybridize to those sequences without the requirement of denaturation.
  • preferred embodiments of the present methods for isothermal strand displacement amplification include an initial step in which a target sequence is analyzed to determine estimated fractions of dissociated bases along the length of the target sequence.
  • the estimated fractions of dissociated bases are calculated by determining enthalpy and entropy for each base in the target sequence using established nearest neighbor dimer values (see SantaLucia, 1998), then using the enthalpy and entropy values to calculate a Tm estimate for each base in the target sequence, then calculating a sigmoidal melt curve growth rate estimate for the target sequence using enthalpy, and then constructing a simulated melt curve to estimate the fraction dissociated for the target sequence at a particular temperature.
  • Primers are then designed to hybridize to those regions of the target sequence having a higher estimated fraction of dissociated bases.
  • at least one primer should be designed to hybridize to those portions of the target sequence having an estimated fraction of dissociated bases of about 0.04 to about 0.2 and preferably in the range of about 0.05 to 0.15.
  • Primers designed to hybridize to these particular sequences are more likely to successfully hybridize to single-stranded DNA, without requiring the use of any artificial methods such as heat to produce denaturation. Thus, these primers work effectively in iSDA methods.
  • primers are designed to hybridize to a target sequence in a region of the target sequence having an estimated fraction of dissociated bases of at least 0.04, and preferably the primers are designed to hybridize in one or more regions of the target sequence that are determined to have the maximized estimated fraction of dissociated bases for that particular target sequence.
  • a set of sequences are constructed that are within the full target sequence and the estimated fractions of dissociated bases are calculated for each subsequence.
  • the enthalpy and entropy values are calculated for each subsequence then used to estimate Tm for the subsequence and a melt curve rate around each base of interest. Then, the average value of the estimated fractions of dissociated bases is calculated for each subsequence.
  • a primer is designed to bind to a target sequence in a region of the target sequence that has a favorable estimated fraction of dissociated bases, preferably higher than 0.04.
  • a variety of methods utilizing isothermal amplification methods are known and can be utilized in conjunction with the methods disclosed herein. These include Strand Displacement (SDA), Exponential amplification (EXPAR), Loop-mediated amplification (LAMP), Transcription-mediated amplification (TMA)/Nucleic acid-based amplification (NASBA), Recombinase polymerase amplification (RPA), Helicase-dependent amplification (HAD), and others (Niemz et al., 2011).
  • SDA Strand Displacement
  • EXPAR Exponential amplification
  • LAMP Loop-mediated amplification
  • TMA Transcription-mediated amplification
  • NASBA Transcription-mediated amplification
  • RPA Recombinase polymerase amplification
  • HAD Helicase-dependent amplification
  • the iSDA methods are performed with digital FCR or in a digital format that allows for the determination of absolute nucleic acid concentration.
  • Digital PCR is an established diagnostic tool (Pohl and Shih (2004); Sedlak and Jerome, Diagn Microbiol Infect Dis., (2013)).
  • Digital PCR (dPCR) is based on a combination of limiting dilution, end-point PCR, and Poisson statistics to determine the absolute measure of nucleic acid concentration (US Pat. No. 6,440,706).
  • the use of short MGB FRET probes in dPCR is disclosed in U.S. Patent No. 9,328,384, incorporated by reference.
  • the iSDA methods are performed using probes that include the abasic spacer.
  • some preferred embodiments for iSDA amplification involve the use of probes (Pleiades probes) such as those described herein and shown in Figure 2. Examples of these probes are disclosed in U.S. Patent No. 7,381,818.
  • Pleiades probes include a MGB and a fluorescent dye attached at 5’ end ’ and a quencher at 3’ end of an oligonucleotide that may also include one or more modified bases.
  • Probes that include an abasic spacer are additional preferred embodiments. Real-time iSDA detection shows strong sample amplification and absence of the signal in the NTC samples for the probes including the abasic spacer compared to other probes.
  • probes including the abasic spacer and an endonuclease that is Endonuclease IV the DNA of all species is exposed to both endogenous and exogenous factors, which damage its chemical structure.
  • the most common lesion that arises in cellular DNA is the loss of a base to generate an abasic site, which is typically referred to as an apurinic or apyrimidinic (AP) site.
  • AP apurinic or apyrimidinic
  • oligonucleotide probe for use in iSDA amplification and detection that have the following structure: wherein 03’ is the 3’ oxygen atom of the 3’-terminal deoxyribose ring of one nucleotide sequence (N n ),
  • 05’ is the 5’ oxygen atom of the 5’ -terminal deoxyribose ring of another nucleotide sequence
  • N is a natural or artificial nucleotide or a nucleoside unit
  • n and m are independently from about 5 to about 15,
  • L 1 and L 2 are independently a fluorophore and a quencher, which are covalently bonded to N n and Nm respectively, and Ap is comprised of the following substructure: wherein R 1 , R 2 are selected from H, substituted or unsubstituted hydroxyl, amine, thiol, (Ci- Cioo)alkyl, (C 1 -C 100 )heteroalkyl, (C 1 -C 100 )alkenyl, (C 1 -C 100 )heteroalkenyl, (C 1 -C 100 )alkynyl, (Ci-Ci ⁇ )heteroalkynyl aryl or heteroaryl; R 3 is selected from substituted or unsubstituted (C 1 -C 100 )alkylene or (C 1 -C 100 )heteroalkylene; optionally, any combination of groups selected from R 1 , R 2 and R 3 forms one or more, saturated or unsaturated, substituted
  • Additional preferred embodiments herein relate to an oligonucleotide probe for use in iSDA amplification and detection that have the following structure: wherein the substituents are defined as above.
  • Additional preferred embodiments relate to a method for iSDA amplification and detection using a probe having the structures above containing the abasic spacer, comprising a step in which the probe is cleaved by an endonuclease, which may be Endonuclease IV.
  • the probe having the structure above is cleaved enzymatically to generate a fluorescent signal.
  • FIG. 25 A shows an example structure of a typical Endonuclease IV probe and enhancer detection system. There is a one base gap in target sequence, between the probe and the enhancer.
  • FIG. 25B shows an exemplary probe containing the abasic spacer as disclosed herein for use in detection similar to FIG.
  • the abasic spacer is included. Preferred embodiments described herein, utilizing post amplification iSDA methods, have shown strong sample amplification compared to the system shown in FIG. 25A, with a low IC signal. In contrast to the system shown in FIG. 25A, in preferred embodiments using the probes with abasic spacers shown above, the probes are digested into two pieces, each piece having a Tm substantially lower than the iSDA reaction temperature, limiting potential interactions. In additional preferred embodiments, Endonuclease IV cleavage of probes including abasic spacers having the structure shown above is coupled to an amplification reaction.
  • the better efficiency of the preferred embodiments described herein, where the probe includes the abasic spacer and is cleaved by Endonuclease IV can likely be explained by the cycling mechanism of the signal generation.
  • the melting temperatures of the products, produced by Endo IV enzyme cleavage are lower than the iSDA reaction temperature.
  • the melting temperature of each portion of the digested probe is 46.0°C, as shown in FIG. 26A.
  • 26B shows a meltcurve analysis of a typical undigested Endonuclease IV probe (“Endo IV”) (SEQ ID NO: 83) compared to an undigested probe containing an abasic spacer (“Ap Probe”) (SEQ ID NO:82), both with a synthetic complement.
  • Endo IV probe has a Tm of 62.0°C and the Ap probe has a Tm of 59.0°C. Therefore, the digested products cannot compete for the binding site with the undigested probe. They dissociate from the target and make it accessible for the intact probe to hybridize, get cleaved and release the fluorescent signal.
  • Preferred abasic spacers may be introduced into the probe during automated oligonucleotide synthesis using a phosphoramidite compound, such as any of the phosphoramidite compounds shown below in Table A.
  • the abasic spacer (Ap) in the structure shown above is one of the spacers shown below in Table B.
  • Preferred embodiments herein relate to methods for iSDA using probes that include an abasic spacer. These methods are improvements upon existing technology utilizing the Endo IV enzyme because they do not require a separate enhancer oligonucleotide and are also improvements upon existing technology for iSDA using the Pleiades probe because they show enhanced sensitivity.
  • iSDA was performed using final concentrations of 3.75 mM MgS0 4 , 50 mM KH2PO4 pH 7.6, 250 nM forward primer, 1 ⁇ reverse primer, 50 nM bumper oligonucleotides, 200 nM probe, 0.2 mM dNTPs, 40 pg/mL BSA, 10 ng genomic DNA, 4U N.BbvCIB and 3.6U Bst DNA polymerase in a total volume of 20 ⁇ L (monoreagent). Twenty microliters of the mono-reagent was introduced in a 96 well PCR plate with 10 ⁇ L of sample nucleic acid.
  • Sample nucleic acid was obtained by extraction with easyMag using NucliSENSE easyMAG extraction reagents (Biomerieux, l’Etoile, Prance).
  • the plate was sealed with MicroAmp ® Optical Adhesive Film (Applied Biosystems, Foster City, CA) and then centrifuged to collect the assay solution in the bottom of the plate well.
  • the assay was then performed in an ABI 7500 DX Fast Block Real-time PCR machine at 48°C for 30 minutes.
  • This example demonstrates the efficient iSDA amplification without denaturation of the Idhl gene from easyMag extracted nucleic acid from cultured S. aureus subsp. aureus COL (gil57650036 :262250-263203).
  • the primer, bumper and probe sequences are shown in Table 1.
  • Table 1 illustrates Idhl oligonucleotide sequences for iSDA amplification. Underlined sequences represents the nicking site for N.BbvCIB. The upper case sequence is Idhl specific, the 5’ -end lower case sequence is non-complementary to the Idhl target, and the pDNA sequence is shown in brackets.
  • Q14 is a hexaethylene glycol linker
  • MGB is a DPI3 minor groove binder
  • FAM fluorescein
  • EDQ is the Eclipse ® dark quencher (quenching range 390-625 nm, maximum absorption 522 nm, Epoch Biosciences, Inc., Bothell, WA).
  • 2 ⁇ L of the iSDA Idhl reaction mixture was diluted in 100 ⁇ L of lateral flow buffer with the formulation 15 mM HEPES (pH 8), 1% Triton X-100, 0.5% BSA, 400 mM NaCl, 0.05% NaNa, and 100 ng/ ⁇ L streptavidin-coated 300 nm diameter blue-dyed polystyrene nanoparticles (Seradyn).
  • a nitrocellulose strip 4 x 25 mm, containing an immobilized pDNA oligo complementary to the pDNA capture probe 6.
  • the pDNA was immobilized via a cross-linked polythymidine tail at a concentration of 120 pmol/cm and a line width of approximately 1 mm. Positive results were visualized easily by the naked eye (as seen in Figure
  • This example illustrates the versatility of the design of primers from mecA gene sequences to allow iSDA amplification without denaturation.
  • Nucleic acid was easyMag extracted from cultured S. aureus subsp. aureus COL.
  • the primer, bumper and probe sequences of Design 1 and 2 are shown below in Table 2.
  • the pDNA sequence is shown in brackets.
  • Table 2 shows Designs 1 and 2 oligonucleotide sequences for mecA amplifications. Underlined sequences represent the nicking site for NLBbvClB, the upper case sequence is mecA specific, the 5’ -end lower case sequence is non-complementary to the mecA target, the pDNA sequence is shown in brackets, A* is Super A (US 7,045,610), and Q14 is a hexaethylene glycol linker.
  • This example demonstrates the use of different polymerases in the real-time iSDA amplification.
  • iSDA amplification was performed as described above using either Bst DNA Polymerase (portion of Bacillus stearothermophilus DNA Polymerase, New England BioLabs Inc., Ipswich, MA) or Bst2.0 WarmStart (an in silico designed homologue of Bacillus stearothermophilus DNA Polymerase I, New England BioLabs Inc.).
  • Bst DNA Polymerase portion of Bacillus stearothermophilus DNA Polymerase, New England BioLabs Inc., Ipswich, MA
  • Bst2.0 WarmStart an in silico designed homologue of Bacillus stearothermophilus DNA Polymerase I, New England BioLabs Inc.
  • the latter enzyme amplified mecA target and is active above 45°C. The results are shown in Figure 5, indicating better performance with the Bst2.0 WarmStart enzyme.
  • This example illustrates the iSDA bi-plexing of Idhl and an internal control (“IC”).
  • IC template contains nonsense, non-specific target DNA fragment in a plasmid vector.
  • control nucleic acid comprises the sequence shown in Table 4 below.
  • oligonucleotide sequences for the amplification of the IC were generated as described above for iSDA amplification. Underlined sequences represent the nicking site for NLBbvClB, the upper case sequence is IC- specific, and the 5 ’-end lower case sequence is non-complementary to the IC target.
  • the same Idhl primers, bumper, capture and detection oligonucleotides (Seq. ID# 1, 24-7, Table 1) were used for the bi-plexing of the Idhl with the IC .
  • the IC primers, bumpers, capture and detection probes sequences are shown in Table 4.
  • iSDA amplification was performed as described above, except that the concentration for both Idhl and the IC primers were 250nM for the limiting primer and SOOnM for excess primer, forward and reverse bumper primers were at SOnM, the chimeric pDNA- DNA probe and biotinylated probe at 200nM each. Each target dilution contained 5000 IC2 copies.
  • the amplification reaction was incubated at 48°C for 30 minutes then it was analyzed by lateral flow analysis as described above. The lateral flow analysis is shown in Figure 7 indicating for this particular assay a lower detection limit of 60 copies.
  • This example illustrates the probe specific iSDA detection and differentiation of S. aureus (BAA-1556, ATCC) and S.epidermidis (12228, ATCC).
  • This example illustrates the iSDA amplification of nucleic acid from the same sample extracted with different methods.
  • a S. aureus sample was extracted using the following extraction methods: a) Extraction with chaotropic salts (8M guanidinium HC1 or 4M guanidinium thiocyanate) , with and without the silica spin column.
  • Bacterial cells (5x10 8 cfu) were extracted according to the procedure described in Molecular Cloning: a laboratory manual, (pages 7-19, 7-24). DNA from each extraction was resuspended in 200 ⁇ L of the TE buffer and divided into two lOO ⁇ L aliquots. One aliquot was set aside for PCR and iSDA analysis, and another one was further purified on QIAmp DNA Mini Kit (Qiagen) spin columns according to the product manual. DNA was eluted in 100 ⁇ L of the elution buffer. b) Phenol/chloroform extraction followed by ethanol precipitation. (Molecular Cloning: a laboratory manual, App.E3-E4). c) Sonication for 10 min in the waterbath sonicator(Branson 5510, Bransonic). d) 10% final concentration of Triton X100 incubation at room temperature followed by ethanol precipitation.
  • This example illustrates the iSDA amplification of the Idhl gene with primers and probes designed with the current disclosure in comparison with traditional designed primers and probes shown in Table 5.
  • the primers and bumper primers for the Idhl gene described in Tables 1 and 5 were tested in which both sets of primers had target concentrations ranging from 5 xlO 3 to 5 xlO 5 target copies/reaction.
  • the amplification reactions were analyzed by agarose gel electrophoresis as shown in Figure 11A and B.
  • the arrows in Figure 11A and B refer to the amplicon products of amplification.
  • the amplification with the primers of the current disclosure showed substantial amplification at all three concentrations, while the conventional designed primers showed poor amplification Figure 11 A.
  • RT-iSDA uses the same final concentrations as disclosed for iSDA in [0049], except that 8U WarmStart Bst Polymerase was substituted for Bst Polymerase, 8U NLBbvCl nicking enzyme was used per 10 ⁇ L reaction.
  • the reaction mixture contains 10U RNA inhibitor (Life Technologies), 0.5 ⁇ L Omniscript Reverse Transcriptase (Qiagne), template RNA and 1 pg BSA per 10 ⁇ L/reaction. Reaction mixture was followed in real-time for 25 minutes at 49°C as illustrated in Figure 12a) and lateral flow detection in Figure 12b).
  • the lateral flow membrane has a test line of pDNA (immobilized by cross-linked polythymidine tail) and a BSA-biotin line as flow control.
  • This example illustrates the iSDA amplification of native and denatured P.falciparum genomic DNA.
  • Primers and probes were designed using mitochondrial DNA (Policy et.al,. J. Clin. Microbiol, 48:2866-2871 (2010)) as a target and is shown in Table 7 below. Extraction from Plasmodium falciparum, strain NF54 and iSDA amplification were performed as described above.
  • Figure 13A shows identical real-time iSDA amplification for native and denature DNA at 95°C for 5 minutes.
  • Figure 13B shows the amplification of native DNA at 100 and 1000 copies.
  • Example 20 illustrate the calculation of estimated fraction of dissociated bases within subregions of the Influenza A virus segment 7 matrix protein 2 (M2) and matrix protein 1 (Ml) genes (GenBank: MF599466.1).
  • Example 20 shows the calculation in greater detail. For each subregion (oligo lengths 5 to 41) over the entire sequence, melt curves were predicted using nearest neighbor thermodynamic parameters (SantaLucia 1998) and salt corrections were made to entropy values (see Owczarzy et al, Biochemistry 2008, 47, 5336-5353). Owczarzy developed equations that obtain corrected Tm for non-standard salt conditions (where 1 M monovalent cation is standard), as shown below:
  • Tm(Na) is the predicted Tm, in Kelvins, of the duplex in an environment that may be a mixture of monovalent and divalent cations
  • Tm(Mg) is the predicted Tm, in Kelvins, of the duplex in an environment that may be a mixture of monovalent and divalent cations
  • Tm(l MNa) is the predicted Tm, in Kelvins, of the duplex in a standard solution containing 1 M monovalent cation, calculated by summing standard nearest neighbor enthalpy and entropy terms;
  • fGC is the fraction of duplex which is either guanidine or cytidine;
  • bp is the length of the duplex;
  • [Mg] represents the concentration of divalent cations
  • [Na] represents the concentration of divalent cations
  • b -9.11e-6 K -1
  • c 6.26e-5 ⁇ -1
  • e -4.82e-4 K -1
  • f 5.25e-4 K -1
  • a, d, g in the second equation vary with the ratio, r, of divalent cation ([Mg]) versus monovalent cation ([Na])
  • This example analyzes the Staphylococcus aureus mecA assay designs described in Example 2 above (design 1 and design 2), with the results shown in FIG. 4.
  • the estimated fraction of bases dissociated within sub-regions of the target gene was calculated using the same process described above in Example 11.
  • the results are shown in FIG. 15.
  • the primers of design 1 and 2 were designed to hybridize to portions of the target gene. As shown, the primers of design 2 hybridize to a gene region where the estimated fraction of dissociation is about 50% greater than that of design 1. Accordingly, the assay design 2 from Example 2 (and FIG. 4) works better than that of design 1.
  • Design 1 shows a Ct of about 15 at 50 copies while design 2 at the same concentration shows a Ct of 8. This can be explained by the fact that primers from design 2 are designed to hybridize to regions having a higher estimated fraction of dissociation.
  • This example illustrates the performance of iSDA amplification of the Idhl Staphylococcus aureus gene in digital format for target quantitation.
  • the reaction formulation of Example 1, which targeted Idh was repeated using 1000, 100, 10, and 0 copies/ ⁇ L and reaction mixes were loaded on an Applied Biosystems QuantStudioTM 3D Digital PCR Chip v2.
  • Isothermal amplification of the digital chips was performed on the Applied Biosystems ProFlex PCR system at 50 °C for 30 minutes, and chips were imaged using Applied Biosystems QuantStudio 3D chip imager.
  • Table 8 below shows the quantitation result of Idh digital iSDA.
  • FIG. 16 shows corrected images of the chip imager.
  • This example illustrates the prospective design of a CMV iSDA assay by estimating fraction dissociated DNA within sub-regions of this gene, followed by primer design in favorable breathing regions.
  • the primer sequences evaluated are shown in Table 9 below, where the nicking site is underlined, the stabilizing flap sequence is shown in lower case, A* is Super A, T* is Super T (US 7,045,610) and the position of the 3’-end is indicated in FIG. 17.
  • Solid arrows in FIG. 17 indicate good amplification by the particular primer also shown with a plus in Table 9. Empty arrows indicate no amplification.
  • This example analyzes the Plasmodium falciparum assay designs described in Example 10, with results shown in FIG. 13. The estimated fraction of bases dissociated within sub-regions of the target gene was calculated. The results are shown in FIG. 18.
  • Each primer used in the assay design hybridizes within the “breathing profile” of the gene, or those regions where there is a higher estimated fraction of dissociated bases.
  • the reverse primer hybridizes to the gene region where the estimated fraction of dissociation is particularly favorable for breathing.
  • This example analyzes the RSV assay designs described in Example 9, with the results shown in FIG. 12.
  • the estimated fraction of dissociated bases within sub-regions of the target gene was calculated.
  • the results are shown in FIG. 19, which also identifies where the primers were designed to hybridize.
  • the reverse primer hybridizes to a gene region where the estimated fraction of dissociation is particularly favorable for breathing.
  • This example analyzes the IC2 assay designs described in Example 5 and Table 4, with particular attention to SEQ ID NO: 36.
  • the estimated fraction of dissociated bases within sub-regions of this sequence was calculated.
  • the results are shown in FIG. 20, which identifies where the designed primers hybridize to SEQ ID NO: 36.
  • the reverse primer hybridizes to a gene region where the estimated fraction of dissociation is particularly favorable for breathing.
  • This example illustrates a primer design based on first calculating the estimated fraction of dissociated bases in an enterovirus target.
  • the favorable design includes SEQ ID NOs 66 and 67
  • the unfavorable design includes SEQ ID NOs 68 and 69.
  • Figure 21 A shows the profile of estimated fractions of dissociated bases, or breathing profile, of the target sequence with primer locations identified.
  • Example 20 shows the gel image, where primers lying in the breathing profile troughs (SEQ ID NOs: 68 and 69) show non-specific side products, while primers in regions with a greater estimated fraction of dissociated bases (SEQ ID NOs: 66 and 67) show more specific products.
  • Example 20 provides a more detailed calculation of the estimated fraction of dissociated bases in an enterovirus target.
  • This example illustrates a primer design based on first calculating the estimated fraction of dissociated bases in an influenza A virus subtype H3N2 target (>A/Bethesda/P0054/2015 IKY487749I01/13/2015 IUS AIMarylandlH3N2.), shown in FIG. 22.
  • the favorable design includes the primer combination SEQ ID NOs 71 and 72
  • the unfavorable design includes the primer combination SEQ ID NOs 70 and 72.
  • FIG. 22 shows the profile of estimated fractions of dissociated bases, or breathing profile, of the target sequence with primer locations identified.
  • FIG. 23A shows the gel image, where primers lying in the breathing profile with one region of lower dissociated bases (SEQ ID NO: 70) combined with a region of higher dissociated bases (SEQ ID NO: 72) show non-specific side products, while primers in regions with a greater estimated fraction of dissociated bases (SEQ ID NOs: 71 and 72) show more specific products.
  • FIG. 23B shows the gel image of a titration of influenza A virus subtype H3N1 from 3 to 300 copies/reaction.
  • FIG.23C shows the gel image of a titration of influenza A vims subtype H3N2 at 50 copies/reaction in the presences of 10 to 100 ng of human genomic DNA, illustrating the robustness of the amplification.
  • Example SEQ ID NO:77 represents an enterovirus (Cocksaclde A16) region used in the exemplary calculation:
  • the first base in SEQ ID NO:77, T is then analyzed by construction of subsequences centered about the first base.
  • Each subsequence in Step 2 is then analyzed for enthalpy and entropy using a dimer table (see Table 13 below for Unified Enthalpy and Entropy Parameters; SantaLucia 1998).
  • Constant c was empirically calculated from model systems to be 365.608, as an average
  • FIG.27 shows the fraction of dissociated bases for each base in the sequence of SEQ ID NO:78. Arrows and lines indicate the sequences chosen for the forward primer, reverse primer, reverse bumper primer, and probes to be used in the assay.
  • the primers and probes are identified in Table 14 below.
  • the “Ap probe” contained an abasic spacer labeled Ap that was Spacer 1 from Table A above inserted between the eighth and ninth bases.
  • the Ap probe used in the assay also contained a quencher which was the Eclipse Dark Quencher, shown in FIG.
  • FIG. 37A attached to the first base and a fluorophore which was fluorescein-labeled uridine, shown in FIG. 37B, attached to the eleventh base, to the right of the abasic spacer.
  • a probe and an enhancer to be used in typical Endonuclease IV detection were also chosen and are identified below in Table 14.
  • the Endo IV probe also contained a quencher which was the Eclipse Dark Quencher attached to the first base and a fluorophore which was fluorescein attached to the eleventh base.
  • the probe sequences are also illustrated in FIG. 25A and FIG. 25B.
  • the lower case bases are noncomplementary to the target sequence
  • the upper case bases are specific to the target sequence
  • the underlined portion is the nicking site for NLBbvClB.
  • nucleic acid extraction and amplification was performed using ELITe InGenius fully automated sample-to-result instrument.
  • iSDA was performed in one step with a reverse transcription reaction, using final concentrations of 8 mM MgSO ⁇ t, 50 mM KH2PO4 pH 7.6, 1 ⁇ forward primer (SEQ ID NO: 79), 1 ⁇ reverse primer (SEQ ID NO: 80), lOOnM reverse bumper (SEQ ID NO: 81), 500 nM Ap Probe (SEQ ID NO: 82) or 250 nM Pleiades probe (SEQ ID NO: 85), 0.2 mM dNTPs, 4U N.BbvClB, 20U Bst 2.0 WarmStart DNA polymerase, 7.5U WarmStart RTx, 5U Endonuclease IV in a total volume of 25 ⁇ L.
  • the final reaction volume was obtained by combining 15 ⁇ L of mixture of iSDA reagents with
  • Detection using Pleiades probe in iSDA reaction shows that there is no detectable signal from the NTC sample with interrogation of high concentration echovirus samples (10 4 copies/reaction). This is shown in FIG. 28 A. However, at lower sample concentrations, such as 100 copies, when intensity of the specific signal significantly drops it is difficult to discriminate between the sample and the NTC signals. This is shown in FIG. 28B. However, use of the Endo IV probe for detection of the samples at low concentration (100 copies/reaction), showed an absence of NTC signal, but also a relatively weak sample signal. This is shown in FIG. 29.
  • FIG. 30 shows a comparison of signal strength between Endo IV probe and the Ap
  • the NTC shows no signal
  • the probe containing the abasic spacer shows a significantly higher signal for the amplified target.
  • FIG. 31 A shows results of amplification using a Pleiades probe (SEQ ID NO: 85) for detection of echovirus at 100 target copies per reaction
  • FIG. 3 IB shows results of amplification of the same amount of target using the Ap probe. While both probes generate similar signal strength, the detections using a Pleiades probe shows a significant NTC signal, while the detection using a probe with the abasic spacer as described herein shows no NTC signal.
  • Another experiment compared the signal generation by the Endo IV probe and the Ap probe in a reaction containing only Endonuclease IV enzyme, iSDA buffer and a synthetic complement template. No other iSDA reagents were included in the reaction. Concentration of each probe was 500 nM and that of the complement was 25 nM. Reaction temperature was 50°C. Ten times stronger signal was observed with the probe containing the abasic spacer, as shown in FIG. 32. A better efficiency of the Ap probe can likely be explained by the cycling mechanism of the signal generation. The melting temperatures of the products, produced by Endo IV enzyme cleavage, are lower than the iSDA reaction temperature. Therefore, these products cannot compete for the binding site with the undigested probe.
  • Potassium phosphate concentration was evaluated at 50, 55 and 58 mM, respectively at Echovirus RNA levels of 5, 20, 25 copies/reaction. Each level was tested in four replicate samples. As shown in Table 15 below, the sensitivity was the highest in the reactions with phosphate concentration at 55 mM.
  • Potassium phosphate concentration also has an effect on side product formation.
  • the formation of the non-specific side products is a common problem in various isothermal amplification reactions, which significantly reduce the sensitivity of the these methods (Niemz et al., Nucleic Acids Res. 40(11): e87 (2012)).
  • the duplicate NTC samples were used instead of RNA in iSDA reactions described above.
  • reactions were mn for 10 and 11 minutes. After the completion of iSDA the samples were analyzed on agarose gel. The results are shown in FIG. 34.
  • potassium phosphate concentration has a significant effect on side product formation with 58mM concentration producing the least amount.
  • Increased potassium phosphate concentration likely reduces formation of the side products by having an inhibitory effect on the enzyme’s activity.
  • target concentration in the reaction is low, the fast accumulating side products effectively compete for resources (primers, enzymes, dNTP) of the iSDA reaction and therefore reduce its sensitivity.
  • the data presented in Table 15 and FIG. 34 support a conclusion that increased potassium phosphate concentration reduces the amount of side product and helps amplification of the target at low copy number.
  • FIG. 35 shows the results of a 10 fold titration of a high positive clinical echovirus sample (approx. 10 6 copies/reaction).
  • iSDA resulted in significantly higher fluorescent signal and was completed in 15 minutes when PCR reaction only finished its reverse transcription step.
  • a similar experiment compared iSDA amplification using Ap probe with PCR at 25, 12, 10 and 5 copies/reaction.

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Abstract

L'invention concerne des procédés, des amorces et des sondes pour l'amplification et la détection isothermes, sans dénaturation, de cibles d'acide nucléique double brin pour l'amplification par déplacement de brin par polymérase (iSDA). Les procédés et les compositions de l'invention sont hautement spécifiques pour des cibles d'acides nucléiques avec une sensibilité, une spécificité et une vitesse élevées permettant la détection de niveaux cibles pertinents cliniques. Les procédés et les compositions peuvent être facilement utilisés pour amplifier ou détecter des cibles d'acide nucléique dans des échantillons biologiques.
PCT/US2019/065210 2019-10-23 2019-12-09 Procédés d'amplification par déplacement de brin isotherme véritable WO2021080629A1 (fr)

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