CN112239776A - Multiple nucleic acid detection method and kit based on hybridization and cascade signal amplification principle - Google Patents
Multiple nucleic acid detection method and kit based on hybridization and cascade signal amplification principle Download PDFInfo
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- CN112239776A CN112239776A CN202011145314.2A CN202011145314A CN112239776A CN 112239776 A CN112239776 A CN 112239776A CN 202011145314 A CN202011145314 A CN 202011145314A CN 112239776 A CN112239776 A CN 112239776A
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- C12Q2600/16—Primer sets for multiplex assays
Abstract
The invention relates to a multiple nucleic acid detection method based on hybridization and cascade signal amplification principles, which can simultaneously detect a plurality of target nucleic acid molecules in a sample. The invention also relates to compositions and kits relating to the method. The invention realizes the specific capture of target nucleic acid molecules by a plurality of groups of pre-coated probes and capture bridging probes, and realizes the multiple detection of the target nucleic acid molecules in a sample by connecting the labeled bridging probes with a cascade signal amplification system, thereby achieving the aim of detecting the gene expression level, and providing a basis for the application of disease pathogenesis elucidation, biomarker screening, diagnosis and prognosis indication, treatment scheme selection and the like.
Description
Technical Field
The present invention relates to the field of biotechnology, in particular, to a method, composition and kit for detecting and/or quantifying a target nucleic acid molecule in a sample. The detection method can simultaneously detect a plurality of target nucleic acid molecules in a sample.
Background
The detection of specific nucleic acid molecules has been widely used for the identification of diseases or disease subtypes, disease course monitoring, prognosis evaluation, and the like. Current common means of nucleic acid detection include: real-time fluorescent quantitative PCR (qPCR) technology, gene chip, gene sequencing and the like. However, these techniques are complicated in operation, time-consuming and labor-consuming, expensive in detection cost, or have high requirements for samples.
At present, the qPCR technology which is most widely applied in clinical medical examination belongs to a template amplification technology, and the template amplification is carried out by utilizing enzymatic reaction, and then the fluorescence detection process is carried out. The technology has high requirement on the integrity of the template, and the reaction is seriously influenced by a PCR inhibitor in a sample, so that the type and the processing process of the sample are strictly limited; in addition, the technology is not suitable for simultaneously detecting a large number of target nucleic acid molecules in one sample due to the limitation of the number of fluorescence detection channels of the instrument and the fluorescent label in the reaction amplification system.
As is well known, formalin-fixed paraffin-embedded (FFPE) tissue samples are widely used in hospitals or tissue sample banks worldwide for histological diagnosis and research due to their long-term normal temperature preservation. These precious and widely available medical research materials provide valuable resources for retrospective research, elucidation of disease pathogenesis, biomarker screening, prognostic indication, etc.; in particular, the clinical outcome of these specimens has been well documented and is therefore the best choice for retrospective study clinical studies. However, high quality nucleic acid is not easy to remain in the FFPE sample, and even though a special nucleic acid extraction process is performed, the qPCR technology still has the problems of low accuracy and poor reproducibility in the quantification of nucleic acid in the FFPE sample.
The invention patent publication CN105087772A discloses a liquid-phase gene chip detection method, which provides a method for amplifying a sample DNA to be detected through a bead-primer conjugate having a primer function to reduce the risk of false positives, but still requires a PCR reaction, resulting in an increase in product cost, and has a problem that it is difficult to detect a small amount of sample or a small amount of nucleic acid contained in the sample, and it is impossible to simultaneously detect a plurality of target nucleic acid molecules with respect to a single sample.
Disclosure of Invention
In order to solve the defects in the nucleic acid detection process in the prior art, the invention provides a multiplex nucleic acid detection method based on the principles of hybridization and cascade signal amplification, which utilizes a plurality of groups of distinguishable microspheres, captures nucleic acid molecules through hybridization of a plurality of groups of specific probe combinations, then performs cascade signal amplification, uses a commercialized detection instrument, can realize multiplex detection of a plurality of target nucleic acid molecules in a single sample in the same hole without PCR reaction, is particularly suitable for high-throughput detection of a plurality of target genes in a large number of samples, and provides a composition and a kit related to the method.
In order to achieve the above purpose, the invention provides the following technical scheme:
in embodiments, the present invention provides a multiplex nucleic acid detection method comprising the steps of:
1) obtaining a sample containing a target nucleic acid molecule;
2) mixing the sample with at least one group of microspheres pre-coated with target nucleic acid molecule specific probes, and adding at least one group of specific probe combinations corresponding to the target nucleic acid molecules, wherein the specific probe combinations comprise capture bridge probes and label bridge probes;
3) adding at least one group of cascade signal amplification systems, wherein the cascade signal amplification systems comprise primary signal amplification molecules, secondary signal amplification molecules and signal probes;
in the detection process, target nucleic acid molecules are subjected to complementary hybridization with the pre-coated probe through the capture bridging probe so as to be immobilized on the microspheres; the target nucleic acid molecule is connected with the cascade signal amplification system through a labeled bridging probe; the signal probe provides a signal to be detected, so that the target nucleic acid molecule is detected, and the target nucleic acid in the sample is detected through the detection system.
Preferably, the microspheres carry distinguishable labels.
More preferably, the distinguishable label is a barcode or a fluorescent dye code.
Preferably, the surface of the microsphere is chemically modified and/or magnetic.
More preferably, the chemical modification of the surface of the microsphere is hydroxyl modification.
Preferably, the probe combination further comprises a shielding probe for shielding some sequence regions on the target nucleic acid molecule, thereby reducing non-specific hybridization.
Preferably, the capture bridge probe, the label bridge probe and the shielding probe each have a region complementary to the target nucleic acid molecule, and the sequence length of the complementary region is 15-30 nucleotides.
Preferably, each set of capture bridge probes comprises 2-10 different capture bridge probes having two sequence regions, one sequence region being a target nucleic acid molecule specific binding region capable of hybridising to a specific sequence region of a target nucleic acid molecule and the other sequence region being a pre-coated probe binding region capable of hybridising to a pre-coated target nucleic acid molecule specific probe.
Preferably, each set of labeled bridge probes comprises 2-10 different labeled bridge probes, each of which has two sequence regions, one being a target nucleic acid molecule specific binding region capable of hybridizing with a specific sequence region of a target nucleic acid molecule, and the other being a cascade signal amplification system binding region capable of hybridizing with a cascade signal amplification system.
Preferably, each set of shielding probes comprises 2-10 different shielding probes capable of hybridizing to regions of the target nucleic acid molecule that are not hybridized to the capture bridge probe, the label bridge probe.
Preferably, the primary signal amplification molecule has two sequence regions, one sequence region being a leader region capable of complementary hybridization with the labeled bridge probe, and the other sequence region being composed of a plurality of repeated sequences capable of hybridization with the primary signal amplification molecule.
Preferably, the secondary signal amplification molecule has two sequence regions, one sequence region being a leader region capable of hybridizing with the primary signal amplification molecule, and the other sequence region being composed of a plurality of repeated sequences capable of hybridizing with the signal probe.
Preferably, the signaling probe has two sequence regions, one sequence region being capable of complementary hybridization to a secondary signal amplification molecule and the other sequence region comprising a reporter molecule that directly or indirectly provides a detectable signal or a label that can bind or react with the reporter molecule.
More preferably, the aforementioned detection system may be an instrument having a fluorescence reading, a chemiluminescence reading, or a radiation reading function.
More preferably, the detection system comprises a computer and detection analysis software.
Preferably, the sample comprises DNA.
Preferably, the sample comprises RNA.
Preferably, the sample is a damaged sample.
More preferably, the damaged sample comprises a paraffin-embedded sample.
Preferably, the at least one set of probes specific to the pre-coated target nucleic acid molecule, the at least one set of specific probe combination, and the at least one set of cascade signal amplification system refer to one or more sets.
In another embodiment, the present invention also provides a composition for use in a multiplex nucleic acid detection method, the composition comprising: at least one group of microspheres pre-coated with target nucleic acid specific probes, at least one group of specific probe combinations and at least one group of cascade signal amplification systems; wherein the specific probe combination comprises a capture bridge probe and a labeled bridge probe; the cascade signal amplification system comprises a primary signal amplification molecule, a secondary signal amplification molecule and a signal probe.
Preferably, the specific probe combination further comprises a shielding probe.
In another embodiment, the present invention also provides a multiplex nucleic acid detection kit, which comprises the above composition.
The process of the method provided by the invention can be divided into the following three steps:
(1) separation of target nucleic acid molecules: for different types of samples, corresponding commercial kits can be used for cracking the samples to release nucleic acid molecules therein, and sample lysates containing target nucleic acid molecules are obtained.
(2) Target nucleic acid molecule capture: and (2) capturing and fixing the target nucleic acid molecule in the sample lysate in the step (1) on the microsphere carrier through complementary hybridization of the target nucleic acid molecule and a pre-coated Probe (PC) coated on the distinguishable microsphere carrier. The probe set generally includes a capture bridge probe (CB), a label bridge probe (LB), and a shield probe (BL).
(3) Signal amplification and detection: and (3) utilizing a cascade signal amplification system to realize the signal amplification process of the target nucleic acid molecules captured in the step (2), and then obtaining the signal intensity through a proper detection instrument to realize qualitative or quantitative analysis of the target nucleic acid molecules in the sample.
Specifically, for N nucleic acid molecules to be detected contained or suspected to be contained in a sample, N groups of optionally distinguishable microsphere carriers are selected, for example, N different fluorescence-encoded microspheres, or N groups of microspheres with different barcodes (N is a positive integer greater than or equal to 2). Each set of microsphere carriers was attached to a different pre-coated probe. In addition, typically, M capture bridge probes are designed for M different sequence regions in each target nucleic acid molecule, which constitute a set of capture bridge probes specific for that target nucleic acid molecule, and which all hybridize complementarily to the same pre-coated probe (M is a positive integer greater than or equal to 2. typically, M does not exceed 10). Therefore, each target nucleic acid molecule in the sample is complementarily hybridized with a group of specific capture bridging probes corresponding to the target nucleic acid molecule, and then the group of capture bridging probes is complementarily hybridized with a corresponding pre-coated probe, so that the one-to-one correspondence of one target nucleic acid molecule and one microsphere is realized.
A plurality of target nucleic acid molecules in a sample are respectively fixed on a plurality of groups of distinguishable microsphere carriers after corresponding capture bridging probes and corresponding pre-coated probes are hybridized. And then, through complementary hybridization of the labeled bridging probe, the target nucleic acid molecule and the cascade signal amplification system, a conjugate in the form of 'fluorescence-encoded fluorescent microsphere-precoated probe-capture bridging probe-target nucleic acid molecule-labeled bridging probe-cascade signal amplification system' is formed. By using a signal detection instrument, qualitative and quantitative detection of the target nucleic acid molecules in the sample can be realized by detecting the label in the cascade signal amplification system on the conjugate and the signal intensity of the label. Meanwhile, through the identification and the distinguishing of the microsphere carrier, the multiple fluorescent microspheres in the sample and the target nucleic acid molecules corresponding to the fluorescent microspheres can be distinguished. Therefore, by the above technical scheme, multiple detection of multiple nucleic acid molecules in a single sample can be realized.
In the above technical solution, the distinguishable microsphere carrier can be any distinguishable solid phase microsphere, generally having a flat surface and having rigidity. Materials for microsphere fabrication include, but are not limited to, glass, silicon, silica, quartz, and polystyrene. The microspheres in each group can be distinguished according to their fluorescence emission spectra, diameter sizes, printed bar codes or combinations thereof. The microspheroidal supports typically have a surface modification to facilitate binding of the pre-coated probes. For example, in some embodiments, the surface of the microsphere is hydroxyl modified, and the amino modified probe can be coated by a chemical reaction. Preferably, the microspheres are magnetic to facilitate separation from the solution.
In the above embodiments, the pre-coated probe is a polynucleotide, and a pre-coated probe typically comprises at least one polynucleotide sequence complementary to a portion of the polynucleotide sequence of the at least one capture bridge probe, the portion of complementary hybridization typically being 15-25 nucleotides in length. The pre-coated probes may be attached to the microspheroidal support covalently or non-covalently, directly or through a linker molecule. For example, in some embodiments, the pre-coated probes may be amino-modified by chemical reactionCan be combined on the microsphere with the surface hydroxyl modified. For example, in some embodiments, the pre-coated probe sequences may include, but are not limited to, sequences of: (5 '→ 3') NH2-ttttttAAACCGAACTCAAAG(SEQ ID NO:1)、NH2-ttttttGAAAACGGTAACTTC(SEQ ID NO:29)、NH2-ttttttAGGCTAACATTTGAA(SEQ ID NO:53)、NH2-ttttttAATGCTTTGACTCAG(SEQ ID NO:79)、NH2ttttttACTTTCTTTCCAAGAG (SEQ ID NO:104), and so forth. Preferably, the pre-coated probes are single-stranded polynucleotides.
In the above technical solution, the capture bridge probe is a polynucleotide, and one capture bridge probe has two sequence regions, wherein one sequence region is a specific binding region of a target nucleic acid molecule and can hybridize with a specific sequence region of the target nucleic acid molecule, and the other sequence region is a binding region of a pre-coated probe and can hybridize with the pre-coated probe. In general, 2 or more (usually no more than 10) capture bridge probes for different sequence regions of the target nucleic acid molecule are designed for the same target nucleic acid molecule, each capture bridge probe has a different specific binding region but the same pre-coated probe binding region, so that multiple capture bridge probes of the same target nucleic acid molecule can be bound to the same microsphere carrier through the pre-coated probe binding regions. Preferably, the capture bridge probe is a single stranded polynucleotide. For example, in some embodiments, the general sequence structure of the capture bridge probe is: (5 '→ 3')
tttttTTCAAATGTTAGCCTWherein, in the step (A),
represents a specific binding region of a target nucleic acid molecule, and needs to be specifically designed according to the target nucleic acid molecule;TTCAAATGTTAGCCTfor pre-coating the probe-binding region, in other embodiments, a pre-coated probe-binding region capable of complementary hybridization with a pre-coated probe can be designed based on the sequence of a different pre-coated probe; ttttttt represents the nucleotide sequence ttttttt for distinguishing the specific junction of the target nucleic acid moleculeSpacer sequences (spacers) between the binding region and the pre-coated probe binding region. In general, the design of capture bridge probes is based on the target nucleic acid sequence and the pre-coated probe sequence.
In the above technical solution, the labeled bridge probe is a polynucleotide, and one labeled bridge probe has two sequence regions, wherein one sequence region is a specific binding region of a target nucleic acid molecule and can hybridize with a specific sequence region of the target nucleic acid molecule, and the other sequence region is a bridge region of a cascade signal amplification system and can hybridize with a primary signal amplification molecule in the cascade signal amplification system. Preferably, the labeled bridging probe is a single stranded polynucleotide. For example, in some embodiments, the general sequence structure of the labeled bridge probe is: (5 '→ 3')
tttttGTATGCGCGCTGCTATGCCGWherein, in the step (A),
represents a specific binding region of a target nucleic acid molecule, and needs to be specifically designed according to the target nucleic acid molecule;GTATGCGCGCTGCTATGCCGthe bridge area of the cascade signal amplification system can be used as a general design; ttttttt represents the nucleotide sequence ttttttt, a spacer (spacer) for distinguishing the specific binding region of the target nucleic acid molecule from the bridge region of the cascade signal amplification system.
In the above technical solution, the shielding probe is a polynucleotide capable of hybridizing with a sequence region of the target nucleic acid molecule that is not hybridized with the capture bridge probe and the label bridge probe, thereby reducing non-specific binding between the target nucleic acid molecule and the probe and reducing background noise of detection. Preferably, the shielding probe is a single stranded polynucleotide.
In the above technical solution, the probe combination consisting of the capture bridge probe, the label bridge probe and the shielding probe is sequence-specific for the target nucleic acid molecule. The capture bridge probe, label bridge probe and shielding probe are complementary to non-overlapping sequence regions on the target nucleic acid molecule, and the arrangement of the regions is typically (but not necessarily) contiguous. Preferably, the capture bridge probe, the label bridge probe and the shield probe are 15-30 nucleotides in length complementary to the target nucleic acid molecule.
In the above technical solution, the cascade signal amplification system comprises a primary signal amplification molecule, a secondary signal amplification molecule and a signal probe, wherein the primary signal amplification molecule can hybridize with the labeled bridge probe and the secondary signal amplification molecule, and the secondary signal amplification molecule hybridizes with the signal probe.
In the above embodiments, the primary signal amplification molecule is a polynucleotide. A primary signal amplification molecule is provided with two sequence regions, wherein one sequence region is a leader region and can be complementarily hybridized with the labeled bridging probe; the other sequence region is a repeat region capable of complementary hybridization to one or more secondary signal amplification molecules. For example, in some embodiments, the primary signal amplification molecule leader sequence is: (5 '→ 3') CGGCATAGCAGCGCGCATAC; the primary signal amplification molecule repeat sequence is: (5 '→ 3') TCCACGGCCCTAGGGACAACG, the number of repetitions being 20. Preferably, the primary signal amplifying molecule is a single stranded polynucleotide.
In the above embodiments, the secondary signal amplification molecule is a polynucleotide. A secondary signal amplification molecule has two sequence regions, one of which is a leader region and comprises at least one polynucleotide sequence capable of complementary hybridization with the primary signal amplification molecule, and the other of which is a repeat region and comprises a plurality of generally, but not necessarily, identical polynucleotide sequences capable of complementary hybridization with the signal probe. For example, in some embodiments, the secondary signal amplification molecule leader sequence is: (5 '→ 3') CGTTGTCCCTAGGGCCGTGGA; the secondary signal amplification molecular repeat sequence is: (5 '→ 3') AGTCAGCGCCGTACCAAGTGC, the number of repetitions being 20. Preferably, the secondary signal amplifying molecule is a single stranded polynucleotide.
In the above technical scheme, a target nucleic acid molecule can amplify a signal 400 times (20 times for a primary signal amplification molecule and 20 times for a secondary signal amplification molecule) under the action of a cascade signal amplification system.
In the above embodiments, the signaling probe is a polynucleotide having two sequence regions, one of which is complementary to a secondary signal amplification molecule and the other of which comprises a reporter molecule that directly or indirectly provides a detectable signal or a label that can bind or react with the reporter molecule. For example, in some embodiments, the signaling probe is directly coupled to a reporter molecule that allows for detection of a signal, including (but not limited to) a fluorescent molecule, a chemiluminescent molecule, a radioactive molecule, and the like. For example, in some embodiments, the label to which the signaling probe is coupled includes, but is not limited to, biotin, horseradish peroxidase, alkaline phosphatase, and the like, and accordingly, the reporter molecule can be a streptavidin-labeled reporter molecule or a substrate for various enzymes. Further, avidin or streptavidin for biotin detection may be labeled with an enzyme (horseradish peroxidase or alkaline phosphatase) having a secondary amplification ability to perform secondary amplification of a signal. For example, in some embodiments, the sequence of the signaling probe is: (5 '→ 3') biotin (dT) -GCACTTGGTACGGCGCTGACT (SEQ ID NO:129), and the reporter molecule is phycoerythrin-modified Streptavidin (SAPE).
In the above technical solution, the detecting device may be a device having the functions of fluorescence reading, chemiluminescence reading, radiation reading, etc. according to the difference of the reporter molecules in the cascade signal amplification system.
In the above technical solution, the capture bridge probe, the label bridge probe, the shielding probe, the primary signal amplification molecule, the secondary signal amplification molecule and the signal probe may (but need not) include chemically modified nucleotides or deoxynucleotides in their sequences. Chemical modifications include, but are not limited to: backbone modifications, base modifications, ribose modifications, bridged nucleic acids, iso-bases, and the like. The capture bridge probe, the labeled bridge probe, the shielding probe, the primary signal amplification molecule, the secondary signal amplification molecule and the signal probe can be obtained by chemical synthesis of commercial companies.
In the above technical solution, in any of the steps, the component immobilized on the microsphere carrier without hybridization may be separated from the component bound to the microsphere carrier. For example, after the capture bridge probe, the labeled bridge probe, the shielding probe, the target nucleic acid molecule in the sample and the pre-coated probe pre-coated on the microsphere carrier are hybridized, the nucleic acid molecules in the capture bridge probe, the labeled bridge probe, the shielding probe and the sample which are not hybridized can be washed and eliminated from the system; after the primary signal amplification molecule, the secondary signal amplification molecule and the signal probe are hybridized with the conjugate obtained in the step (2), the primary signal amplification molecule, the secondary signal amplification molecule and the signal probe which are not hybridized can be washed and eliminated from the system; in step (3) above, in some embodiments, the reporter molecule that has not been hybridized can be cleared from the system by washing after hybridization of the reporter molecule.
In order to realize multiplex nucleic acid detection on a single sample, the invention designs a multi-component detection scheme, which comprises the following steps: the kit comprises a sample containing or suspected of containing N nucleic acid molecules to be detected, N groups of optionally distinguishable microsphere carriers, N groups of pre-coated probes, N groups of capture bridging probes, N groups of labeled bridging probes, a cascade signal amplification system and a signal detection system. Optionally, a shielding probe is also included.
In a preferred technical scheme under the technical scheme, the distinguishable fluorescence-encoded fluorescent microspheres are MagPlex microspheres of the U.S. Luminex company, and are characterized in that the microspheres have the diameter of 5.6 microns and magnetism, are subjected to fluorescence encoding by two fluorescent dyes in different proportions, and have hydroxyl groups on the surfaces. The detection system is MAGPIX or Luminex 100/200 from Luminex corporation in the United states or Bio-Plex 200 from Bio-Rad corporation.
In a preferred embodiment of the above technical solution, the cascade signal amplification system comprises a primary signal amplification molecule, a secondary signal amplification molecule, a signal probe coupled to a signal amplification label, and a reporter molecule. Wherein each primary signal amplification molecule has 20 repeat sequence regions and each secondary signal amplification molecule has 20 repeat sequence regions; the signal amplification marker coupled with the signal probe is biotin, and each signal probe is provided with 1 biotin molecule; the reporter molecule of the signal amplification label is phycoerythrin-labeled Streptavidin (SAPE).
Furthermore, in order to reduce the background noise and false positive result of the detection system and improve the accuracy and sensitivity of the system, in the above technical scheme, 5% to 50% of the iso-bases can be added when designing the primary signal amplification molecule, the secondary signal amplification molecule and the signal probe, specifically, the iso-bases are isocytosine (isoC) and isoguanylic acid (isoG). Preferably, the proportion of the iso-bases is 20 to 30%.
The invention also provides a kit and a detection system for realizing the aim of the invention, namely, the multiplex detection of a plurality of target nucleic acid molecules in a single sample in a single hole. The kit comprises: n groups of microsphere carriers which can be arbitrarily distinguished, a specific probe combination (comprising N groups of pre-coated probes, N groups of capture bridging probes and N groups of label bridging probes), a cascade signal amplification system and a signal detection system. Optionally, a shielding probe is also included. In addition, the kit optionally includes a sample lysate, lysis buffer, diluent, hybridization buffer, and/or wash buffer, as well as one or more nucleic acid standards of known concentration. The kit of the present invention may further contain, as necessary, a tube, a microplate, a test strip for mixing the components, and instruction data for describing the method of use. The detection system may be an instrument with the functions of fluorescence reading, chemiluminescence reading, radiation reading, etc., depending on the reporter molecule in the cascade signal amplification system. Optionally, the detection system further comprises a computer and detection analysis software.
The multiplex nucleic acid detection method of the present patent can be applied to various fields such as genetic analysis, gene expression analysis, cancer or disease detection, forensic analysis (including paternity testing or criminal investigation), surgical transplantation screening, quality control and certification for products or processes, and the like.
Definition of
Probe needle
The term "probe" as used herein denotes a polynucleotide, preferably a single-stranded deoxyribonucleotide (deoxyribonucleotide), including natural (naturally occuring) dnmps (dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides, and may further comprise a ribonucleotide (ribonucleotide).
When a polynucleotide is represented by a letter sequence (e.g., "AGGCTAACATTTGAA"), it is understood that the nucleotides are 5 'to 3' from left to right, or are clearly determinable by one of skill in the art from the context, unless otherwise specified.
Complementary to each other
"complementary" as used herein refers to sufficient complementarity for a probe to selectively hybridize to a target nucleic acid sequence under hybridization conditions, and has the meaning of including both substantial complementarity (substentiality complementarity) and perfect complementarity (perfect complementarity), preferably being perfect complementarity. The term "substantially complementary sequence" used herein includes not only a completely identical sequence but also a sequence that can function as a probe for a specific target sequence and is partially not identical to a sequence to be compared.
The sequence of the probe does not need to have a sequence completely complementary to a part of the sequence of the template, and may have sufficient complementarity within a range that can hybridize with the template and exert its inherent action. Therefore, the probe of the present invention does not need to have a sequence completely complementary to the nucleotide sequence as a template, and may have sufficient complementarity within a range that can hybridize with the template and exert its inherent action. The design of the probe is well within the skill of those skilled in the art, and a primer design program can be used, for example.
Sample(s)
A "sample" as used herein may be any sample that contains or may contain a target nucleic acid molecule and may include at least one cell, cell culture, tissue sample, lysate, extract, solution, or reaction mixture suspected of containing the target nucleic acid. In some embodiments, the sample can be derived from an animal, human, plant, cultured cell line, virus, bacteria, fungus, and the like. In other non-limiting embodiments, the sample can be a cell lysate, an intercellular fluid, a bodily fluid (including, but not limited to, blood, serum, saliva, urine, feces, throat or nasal swab, spinal fluid, lymph, peritoneal fluid, vitreous fluid, tears, semen, vaginal secretions, lung volume fluid, serosal fluid, bronchoalveolar lavage fluid, etc.), a cell culture fluid, and the like. In other embodiments, the sample may be derived from a fresh tissue sample, cryopreserved tissue, biopsy sample, FFPE sample, or the like.
As used herein, a "compromised sample" refers to a sample having degraded nucleic acids. The damaged sample may be due to exposure to physical forces (e.g., shear forces), harsh environments (e.g., heat or ultraviolet light), chemical degradation, biodegradation, or purification separation techniques, among other factors, which degrade nucleic acids with breaks, gaps, or chemical modifications as compared to the normal form of nucleic acids in the natural environment.
Standard preservation techniques for preserving biological tissue samples are typically fixed using formalin, formaldehyde or paraformaldehyde, embedded with paraffin, acrylamide or cellulose. Some preservation processes use high temperatures, such as paraffin infiltration, which can damage the sample, resulting in chemical modification of DNA and RNA. In formalin-fixed and paraffin-embedded (FFPE) samples, RNA or DNA often contains nucleotide-nucleotide and nucleotide-protein cross-links, as well as other chemical modifications that affect Nucleic acid integrity, such as reactions between formaldehyde and nucleotides that form methylene bridges between the amino groups of two nucleotides, which disrupts reverse transcription reactions (Masuda et al, Nucleic Acids Res.27 (22): 4436-4443, 1999). Furthermore, over time, nucleic acids in FFPE samples often continue to degrade, resulting in fragmentation of nucleic acids, particularly RNA. These modifications of DNA and RNA can affect the steps of copying template sequences in traditional polymerase chain reactions, leading to inaccurate measurements and unreliable data.
Target nucleic acid molecule
The "target nucleic acid molecule" as referred to herein comprises the specific nucleic acid sequence to be detected and may be any desired nucleic acid molecule. The source of the nucleic acid may be animal, human, plant, cultured cell line, virus, bacteria, fungi, etc. The target nucleic acid molecule may be single-or double-stranded DNA, DNA: RNA hybrids or RNAs, including but not limited to rRNA, tRNA, stRNA, snRNA, short interfering RNA (sirna), messenger RNA (mrna), small RNA (microrna), long non-coding RNA (incrna), etc., the "target" portion may be double-stranded, single-stranded, or overlap with both double-stranded and single-stranded portions.
Advantageous effects
The invention provides a multiple nucleic acid detection method based on hybridization and cascade signal amplification principles, which utilizes a plurality of groups of distinguishable microspheres to capture nucleic acid molecules through hybridization of a plurality of groups of specific probe combinations, and then detects after cascade signal amplification, can realize multiple detection of a plurality of target nucleic acid molecules in a single sample in the same hole on the premise of not carrying out PCR reaction, and can detect a small amount of samples or a small amount of nucleic acid contained in the samples, thereby realizing the purposes of saving cost and reducing false positive.
Drawings
FIG. 1 is a schematic diagram of the principle of the method of the present invention, showing the composition of a microsphere-specific probe combination-target nucleic acid-cascade signal amplification system complex.
FIGS. 2A-2C are schematic diagrams of combinations of nucleic acid-specific probes according to the methods of the present invention. FIG. 2A shows an example of a target nucleic acid molecule 1, wherein a plurality of capture bridge probes, a plurality of label bridge probes, and a plurality of shielding probes are designed; FIG. 2B is a schematic diagram of a labeled bridge probe comprising a binding region specific for a target nucleic acid molecule and a binding region for a primary signal amplification molecule; FIG. 2C is a schematic diagram of a capture bridge probe comprising a target nucleic acid molecule-specific binding region and a pre-coated probe binding region.
FIGS. 3A-3C are schematic diagrams of a cascade signal amplification system according to the method of the present invention. FIG. 3A is a schematic representation of the combination of a primary signal amplification molecule and a secondary signal amplification molecule, wherein the primary signal amplification molecule comprises a primary signal amplification molecule leader region and a plurality of repeat sequences; FIG. 3B is a schematic representation of the secondary signal amplification molecule and signaling probe combination, wherein the secondary signal amplification molecule comprises a secondary signal amplification molecule leader and a plurality of repeat sequences; FIG. 3C is a schematic of a signaling probe with a sequence complementary to a repeat sequence of a secondary signal amplification molecule and a signal amplification label or reporter.
FIG. 4, relationship between housekeeping gene signal intensity (Net MFI) and RNA concentration.
Target nucleic acid molecule: 1. 10, microspheres: 2. 11, pre-coating probes: 3. 12, capture bridge probe: 4(4-1, 4-2), 13, capture bridge probe target nucleic acid molecule-specific binding region: 4-2-2, pre-coating the capture bridge probe on a probe binding region: 4-2-1, labeled bridging probe: 5(5-1, 5-2), 14, labeled bridge probe target nucleic acid molecule specific binding region: 5-1-1, labeled bridging probe primary signal amplification molecule binding region: 5-1-2, shielding probe: 6(6-1, 6-2), 15, primary signal amplification molecule: 7, secondary signal amplification molecule: 8, signal probe: 9.
Detailed Description
The present invention will be further described with reference to the following embodiments and drawings, and the present invention is not limited to the following embodiments. It is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It is intended that all such alterations and advantages be included in the invention, which occur to those skilled in the art, be considered as within the spirit and scope of the inventive concept, and that all such modifications and advantages be considered as within the scope of the appended claims and any equivalents thereof. In the description and claims of the present invention, the singular forms "a", "an" and "the" include the plural forms unless the context clearly dictates otherwise, e.g., "at least one set of specific probes" means that there may be one and more than one set of oligonucleotide probes. The experimental procedures, without specific conditions being noted in the following examples, are all common knowledge and general knowledge of those skilled in the art, and include various substitutions, modifications/modifications and equivalents known to those skilled in the art, or according to the conditions suggested by the manufacturer. All materials and reagents used in the examples are commercially available products unless otherwise specified.
As shown in FIG. 1, two groups of distinguishable microspheres 2 and 11 are selected for a target nucleic acid molecule 1 to be detected and a target nucleic acid molecule 10 to be detected, wherein the microspheres 2 are combined with pre-coated probes 3, and the microspheres 11 are combined with pre-coated probes 12. A group of specific probe combinations was designed for different sequence regions of the nucleic acid molecule 1: capture bridge probe 4, label bridge probe 5 and shield probe 6. The target nucleic acid molecule 1 is complementarily hybridized with the pre-coated probe 3 through the capture bridge probe 4, and thus immobilized on the microsphere 2. Similarly, the target nucleic acid molecule is immobilized on the microsphere 11 by a factor of 10. Since the microspheres 2 and 11 are distinguishable, the target nucleic acid molecules 1 and 10 can also be distinguished by detecting different labels on the microspheres 2 and 11, respectively.
The target nucleic acid molecule 1 is connected with a cascade signal amplification system through a labeled bridge probe 5. The signal amplification system comprises a primary signal amplification molecule 7, a secondary signal amplification molecule 8, a signal probe 9 and the like. The target nucleic acid molecule 1 can be quantified by detecting the signal intensity of the signal amplification label or the reporter molecule carried by the signal probe 9. Similarly, the target nucleic acid molecule 10 can be quantified.
The shielding probes 6 and 15 can shield some sequence regions on the target nucleic acid molecules, thereby reducing non-specific hybridization and improving the specificity of the whole detection system.
FIG. 2 further illustrates the principle of capturing a target nucleic acid molecule with the specific probe combination according to the present invention. Taking the target nucleic acid molecule 1 as an example, a plurality of capture bridge probes (4-1, 4-2), a plurality of label bridge probes (5-1, 5-2) and a plurality of shielding probes (6-1, 6-2) are designed, so that the specificity and the sensitivity of the system are further improved. Wherein each capture bridge probe (taking 4-2 as an example) comprises a target nucleic acid molecule specific binding region (4-2-2) and a pre-coating probe binding region (4-2-1); each labeled bridge probe (exemplified by 5-1) comprises a target nucleic acid molecule-specific binding region (5-1-1) and a primary signal amplifying molecule binding region (5-1-2). Multiple shielding probes (6-1 and 6-2) can further reduce non-specific binding.
Fig. 3 further illustrates the principle of signal amplification of the cascaded signal amplification system according to the present invention. The cascade signal amplification system comprises a primary signal amplification molecule 7, a secondary signal amplification molecule 8 and a signal probe 9. Wherein each primary signal amplification molecule comprises a primary signal amplification molecule leader region 7-1 and a plurality of repeat sequences 7-2, 7-1 that can hybridize to the primary signal amplification molecule binding region (e.g., 5-1-2) of the labeled bridge probe, and 7-2 that can hybridize to the secondary signal amplification molecule leader region 8-1. Each secondary signal amplification molecule comprises a secondary signal amplification molecule leader 8-1 and a plurality of repeat sequences 8-2, 8-1, which hybridize to the primary signal amplification molecule repeat sequence (7-2), 8-2, which hybridizes to a signaling probe 9. Each signaling probe 9 carries a sequence 9-1 complementary to 8-2 and a signal amplification label or reporter 9-2. FIG. 3A, B shows primary and secondary signal amplification molecules with 20 repeats each, so that the signal can be amplified 400-fold.
Example 1: preparation of 5 housekeeping gene detection kits (HK5)
Using 5 housekeeping genes (RPLP0, GAPDH, ACTB, GUSB, TFRC) as an example, a kit for detecting the above 5 genes was designed and prepared.
The kit comprises the following components: the kit comprises a microsphere carrier pre-coated with probes, a specific probe combination, a primary signal amplification molecule, a secondary signal amplification molecule, a signal probe, a reporter molecule, a lysis solution, a diluent, a washing solution, a standard substance and the like.
The information on the 5 housekeeping genes used in this example is shown in Table 1.
TABLE 1, 5 information about housekeeping genes
| Name of Gene | NM number | Microsphere numbering |
| RPLP0 | NM_001002.4 | 35 |
| GAPDH | NM_002046.7 | 36 |
| ACTB | NM_001101.5 | 47 |
| GUSB | NM_000181.4 | 38 |
| TFRC | NM_003234.4 | 61 |
(1) Designing a probe: the probe combinations for 5 housekeeping genes were designed according to the characteristics of each probe described in the present invention, and are shown in Table 2.
TABLE 2 Probe combination information of 5 housekeeping genes
PC: pre-coated probes on a microsphere carrier, CB: capture bridge probe, LB: labeled bridge probe, BL: the probe is shielded.
(2) Preparation of specific probe set: the probes listed in Table 2 were synthesized by commercial Gene Synthesis and mixed by dissolving with 1 XTE to give final concentrations of 50fmol/L, 200fmol/L and 100fmol/L for each of CB, LB and BL.
(3) Preparation of microsphere group: the surface hydroxyl modified MagPlex microspheres from Luminex corporation, 5 types of which are numbered 35, 36, 47, 38 and 61 respectively, are selected. Each microsphere was washed with 1 XPBS and treated with 0.1M MES (2-morpholinoethanesulfonic acid) (pH 4.5). After removal of the MES solution, every 5X 106The microspheres were coated with 0.5nmol of the corresponding pre-coated probe in 2mg/mL 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride solution protected from light for 4 hours. After the coating, the coating was blocked by adding 10% BSA (bovine serum albumin), washed with 1 XTBS and stored in 1 XTE solution for use. The 5 microspheres were mixed in equal proportions to give groups of HK5 microspheres at a final concentration of 2000 microspheres/μ L each.
Wherein, the 1 × TE solution comprises the following components: 10mM Tris-HCl (pH 7.5-8.0), 1mM EDTA (pH 7.5-8.0).
(4) Design of primary signal amplification molecule and secondary signal amplification molecule:
primary Signal amplification molecule (SEQ ID NO: 127):
secondary signal amplification molecule (SEQ ID NO: 128):
(5) design of the signaling probe:
Biotin(dT)-GCACTTGGTACGGCGCTGACT。(SEQ ID NO:129)
(6) a reporter molecule: SAPE (phycoerythrin-labeled streptavidin) at 20. mu.g/mL.
(7) Preparing a buffer system (the following solution concentrations are final concentrations):
lysis solution: 1M HEPES [4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid ] (pH 8.0), 10% lithium lauryl sulfate, 0.25M EDTA (ethylenediaminetetraacetic acid), 5M lithium chloride, 600mg/l proteinase K.
Washing liquid: 0.1 XSSC, 0.1% SDS (sodium dodecyl sulfate).
Secondary signal amplification molecule dilution: 50% horse serum, 0.5mg/ml proteinase K, 5 XSSC, 1.3% SDS, 6mM Tris-HCl, incubation at 65 ℃ for 2 hours followed by the addition of 6mM phenylmethylsulfonyl fluoride, 0.05% sodium azide, 0.05% ProClin 300 and 10% dextran sulfate.
Standard dilution: 133mM HEPES (pH 7.5), 532mM lithium chloride, 10.6mM EDTA, 1.3% lithium lauryl sulfate, 16mg/ml salmon sperm DNA, 0.04% sodium azide, 0.04% Proclin 300.
Sealing liquid: 10% BSA.
SAPE diluent: 3M tetramethylammonium chloride, 0.01% sodium lauroyl sarcosinate, 50mM Tris (Tris hydroxymethyl aminomethane), 4mM EDTA.
Wherein, the 20 XSSC buffer solution comprises: 3M sodium chloride, 0.3M sodium citrate, pH 6.9-7.1.
Generally, the method of use of the kit described above is as follows:
step 1: the microsphere working solution was prepared according to the following table, protected from light and left at room temperature.
TABLE 3 working fluid compounding Table
Step 2: for cell samples or blood samples, 20 μ L of microsphere working solution and 80 μ L of sample are added to each well; for the FFPE samples, 60. mu.L of microsphere working fluid was added to each well, 40. mu.L of sample. After sealing, incubation was carried out at 600rpm and 54 ℃ for 18-22 hours.
And step 3: the beads were adsorbed for 1 min using a 96-well plate magnetic frame, then the plate wells were discarded and the plate was washed 3 times with 200. mu.L of wash solution.
And 4, step 4: mu.L (0.8fmol) of primary signal amplification molecule solution was added to each well and incubated at 50 ℃ for 1 hour at 600rpm after the plate was closed.
And 5: the beads were adsorbed for 1 min using a 96-well plate magnetic frame, then the plate wells were discarded and the plate was washed 3 times with 200. mu.L of wash solution.
Step 6: add 100. mu.L (1fmol) of secondary signal-amplifying molecule solution to each well and incubate at 600rpm for 1 hour at 50 ℃ after sealing the plate.
And 7: the beads were adsorbed for 1 min using a 96-well plate magnetic frame, then the plate wells were discarded and the plate was washed 3 times with 200. mu.L of wash solution.
And 8: mu.L (0.4fmol) signal probe was added to each well and incubated for 1 hour at 50 ℃ at 600rpm after sealing the plate.
And step 9: the beads were adsorbed for 1 min using a 96-well plate magnetic frame, then the plate wells were discarded and the plate was washed 3 times with 200. mu.L of wash solution.
Step 10: add 100. mu.L of reporter SAPE to each well, seal the plate, 600rpm, and incubate for 30 minutes at room temperature.
Step 11: the beads were adsorbed for 1 min using a 96-well plate magnetic frame, then the plate wells were discarded and the plate was washed 3 times with 2 μ L of wash solution.
Step 12: 130. mu.L of Wash solution 2 was added to each well, shaken at 800rpm for 3 minutes at room temperature, and then the signal intensity was read using Luminex 100/200 or Bio-Plex 200 system.
Example 2: sensitivity, linear dynamic range, accuracy of the microsphere-suspended chip approach
Using 5 housekeeping gene (PRLP0, GAPDH, ACTB, GUSB, TFRC) standards synthesized by Gene Synthesis, the performance parameters of the microsphere suspension chip technology, such as sensitivity, linear dynamic range, accuracy and specificity, were tested using the HK5 detection kit designed in example 1. The results are shown in Table 4.
TABLE 4 detection of Performance parameters of 5 housekeeping genes by the microsphere suspension chip method
(1) Sensitivity and linear dynamic range: 5 housekeeping gene (PRLP0, GAPDH, ACTB, GUSB, TFRC) standards were mixed in equal amounts and diluted in 3-fold gradients to give 10 gradient standard dilutions, ranging in units of amol/. mu.L: 240.00, 80.00, 26.67, 8.89, 2.96, 0.99, 0.33, 0.11, 0.04 and 0.01, and are sequentially marked as Std1, Std2, Std3, Std4, Std5, Std6, Std7, Std8, Std9 and Std 10.
The 10 graded concentrations of standard dilutions and 1 blank were tested using the HK5 test kit designed in example 1. The relationship between signal intensity (Net MFI) and RNA concentration for each housekeeping gene is shown in FIG. 4. The dynamic linear range is about 0.11-120 amol/mu L, and the linear correlation coefficient is 0.997. Meanwhile, the LOD detected by 5 housekeeping genes was 0.04 amol/. mu.L.
In addition, 5 housekeeping genes (PRLP0, GAPDH, ACTB, GUSB, TFRC) had mean background intensities (MFI) of 5.9, 6.8, 5.1, 4.6 and 5.8, respectively, as determined by signal intensity against blank controls (sample dilutions).
(2) Precision: when the sensitivity and linear dynamic range experiments are carried out, repeated experiments of 3 multiple holes are carried out on each of 3 pieces of 96 micro-porous plates. The CV in the plate calculated by the test is less than 10 percent, and the CV between the plates is less than 15 percent, which shows that the method has good precision.
Example 3: detection of 5 housekeeping genes in FFPE samples of ovarian cancer with different storage times
In this example, FFPE samples of ovarian cancer with different storage times were tested, and the expression levels of 5 housekeeping genes (PRLP0, GAPDH, ACTB, GUSB, TFRC) were examined in a total of 198 cases. Sample information is shown in table 5.
Meanwhile, in order to verify the flexibility and accuracy of the nucleic acid detection of the present invention, the above 5 housekeeping genes represent genes with different relative expression levels in the conventional tissues, respectively. The relative expression levels of ACTB, GAPDH and PRLP0 were high, the relative expression level of TFRC was moderate, and the relative expression level of GUSB was low.
The 198 samples were stage II in the tumor stage. Each FFPE specimen was sectioned by a microtome, and it was confirmed by HE staining that the area of the sectioned tumor tissue was more than 70%, the number of sections per specimen was 10, and the total section thickness was 50 μm. After the tissue slices are subjected to digestion treatment and centrifugation by FFPE tissue lysate, the supernatant is taken for a nucleic acid detection experiment, and the detection kit is the HK5 detection kit designed in the embodiment 1. The results are shown in Table 6.
TABLE 5 FFPE sample information Table for ovarian cancer
| Retention time | Number of |
| 5 years old | 57 |
| For 10 years | 50 |
| 15 years old | 50 |
| 20 years old | 41 |
| Total up to | 198 |
TABLE 6 detection results of 5 housekeeping genes in FFPE samples for ovarian cancer
Experimental results show that the method can well distinguish the genes with different relative expression amounts in the FFPE tissue sample. In addition, even for a gene (e.g., GUSB) with a relatively low expression level in an FFPE tissue sample stored for 20 years, the expression level can be accurately measured by the method of the present invention.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Berkeley Nanjing medical research Limited liability company
<120> a multiple nucleic acid detection method and kit based on hybridization and cascade signal amplification principle
<160> 129
<170> SIPOSequenceListing 1.0
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ccagcccaaa ggagaagggg tttttctttg agttcggttt 40
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gggcagcagc cacaaaggca tttttctttg agttcggttt 40
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gacttcacat ggggcaatgg catttttcgg catagcagcg cgcatac 47
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cttctcgggc ccgagaccat ttttcggcat agcagcgcgc atac 44
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gcacatcact caggatttca atggtttttt cggcatagca gcgcgcatac 50
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cacttcaggg ttgtagatgc tgctttttcg gcatagcagc gcgcatac 48
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ccagcacggg cagcagct 18
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gtgttctggg ctggcacagt 20
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acctaaagcc tggaaaaagg aggt 24
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gcccctggag attttagtgg tgat 24
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ctttgtctcc agtcttgatc agct 24
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gtggcttcgc tggctccca 19
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cattgtcgaa cacctgctgg atga 24
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gcagagtttc ctctgtgata tcaag 25
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gatgatagaa tggggtactg atgcaa 26
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ccaggactcg tttgtacccg tt 22
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gaccttttca gcaagtggga aggt 24
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gatggatcag ccaagaaggc ctt 23
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cttcaacctt agctggggct g 21
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ttggcaccaa gccagcgaag tttttgaagt taccgttttc 40
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ggccacagac cacatcacga tttttgaagt taccgttttc 40
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cagatcacat ccacatacgg atttttgaag ttaccgtttt c 41
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gtgataccaa gagtagtagc tgttcaaatt tttgaagtta ccgttttc 48
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tggccagctg cagctgaatc tttttgaagt taccgttttc 40
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cccatagcgg tcacacatct gtttttcggc atagcagcgc gcatac 46
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ggtgggacgc aggctcgttt ttttcggcat agcagcgcgc atac 44
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aactccaggt gcccgtagtc tttttcggca tagcagcgcg catac 45
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cgctctgaat aatgggcttc tgatattttt cggcatagca gcgcgcatac 50
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ccgcggggtg gttcttgtc 19
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cttcaagtag tagccagcag attcta 26
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ggatttggtg tgagcgatca ccat 24
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gcccccttgt ctgctgcata 20
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tgaccaggcg cccaatacga tttttttcaa atgttagcct 40
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catgtagttg aggtcaatga agggtttttt tcaaatgtta gcct 44
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cagcatcgcc ccacttgatt ttgttttttt caaatgttag cct 43
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cttttggctc ccccctgcaa attttttttc aaatgttagc ct 42
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agggggcaga gatgatgacc tttttttcaa atgttagcct 40
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tggtggtgca ggaggcattg tttttttcaa atgttagcct 40
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gatggtgatg ggatttccat tgatgatttt tcggcatagc agcgcgcata c 51
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ctccacgacg tactcagcgc tttttcggca tagcagcgcg catac 45
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gagccccagc cttctccatg tttttcggca tagcagcgcg catac 45
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cttctcatgg ttcacaccca tgactttttc ggcatagcag cgcgcatac 49
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ctgtggtcat gagtccttcc actttttcgg catagcagcg cgcatac 47
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cacgccacag tttcccggag tttttcggca tagcagcgcg catac 45
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ccagagttaa aagcagccct gg 22
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gtcattgatg gcaacaatat ccacttta 28
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gggtggaatc atattggaac atgtaaac 28
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gacggtgcca tggaatttgc cat 23
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gagggatctc gctcctggaa 20
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gtggtgaaga cgccagtgga 20
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ctgatgatct tgaggctgtt gtcata 26
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ggccaggggt gctaagcagt 20
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gataccaaag ttgtcatgga tgacctt 27
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ggtggcagtg atggcatgga 20
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gggccatcca cagtcttctg 20
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gagagccccg cggccat 17
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gtagaggcag ggatgatgtt ctg 23
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ttttttaatg ctttgactca g 21
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caagaaaggg tgtaacgcaa ctttttctga gtcaaagcat t 41
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caaaaaaaaa aaaaaaacca aaacaaaaca aaaaaaattt ttctgagtca aagcatt 57
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ggacaaaaaa gggggaaggg gtttttctga gtcaaagcat t 41
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ccactcccag ggagaccaaa tttttctgag tcaaagcatt 40
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ggtaagccct ggctgcctcc actttttctg agtcaaagca tt 42
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<213> Artificial Sequence (Artificial Sequence)
<400> 85
ggccggactc gtcatactcc tttttcggca tagcagcgcg catac 45
<210> 86
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
gttccagttt ttaaatcctg agtcaagctt tttcggcata gcagcgcgca tac 53
<210> 87
<211> 44
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
gggggatgct cgctccaact ttttcggcat agcagcgcgc atac 44
<210> 88
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
gaatgactat taaaaaaaca acaatgtgca atcaaatttt tcggcatagc agcgcgcata 60
c 61
<210> 89
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
gtggctttta ggatggcaag ggatttttcg gcatagcagc gcgcatac 48
<210> 90
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
caatgctatc acctcccctg tgttttttcg gcatagcagc gcgcatac 48
<210> 91
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
tttgcggtgg acgatggagg 20
<210> 92
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
taagtcatag tccgcctaga agca 24
<210> 93
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
cttgttttct gcgcaagtta ggttttgt 28
<210> 94
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
caaataaagc catgccaatc tcat 24
<210> 95
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
cgactgctgt caccttcacc 20
<210> 96
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
gtcctcggcc acattgtgaa cttt 24
<210> 97
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
cttcctgtaa caacgcatct catatttg 28
<210> 98
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
ccattctcct tagagagaag tggg 24
<210> 99
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
ggacttggga gaggactggg 20
<210> 100
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
gattaaaaaa attttgcatt acataattta cacgaaag 38
<210> 101
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
aataaaacaa aataaaaaag tattaaggcg aa 32
<210> 102
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
gggcacgaag gctcatcatt caa 23
<210> 103
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
agccttcata catctcaagt tggg 24
<210> 104
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
ttttttactt tctttccaag ag 22
<210> 105
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
tcaagaaaaa gacgatcaca gcaatagttt tttctcttgg aaagaaagt 49
<210> 106
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
gcccaagtag ccaatcataa atccaatttt tctcttggaa agaaagt 47
<210> 107
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
cccaatataa gcgacgtgct gctttttctc ttggaaagaa agt 43
<210> 108
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
tatgatcacc gagttttgag cgcttttttc tcttggaaag aaagt 45
<210> 109
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
ccctgctctg acaatcacta tttttctctt ggaaagaaag t 41
<210> 110
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
cctcacgagg gacatatgaa ttttcatttt tttcggcata gcagcgcgca tac 53
<210> 111
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
cacgccagac tttgctgagt ttaaattttt ttcggcatag cagcgcgcat ac 52
<210> 112
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
acccccagga ttctccacca tttttcggca tagcagcgcg catac 45
<210> 113
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
tagatccatt cacaggagtg tataaatctt tttcggcata gcagcgcgca tac 53
<210> 114
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
acaccaattg catttaagct ttcagcattt tttcggcata gcagcgcgca tac 53
<210> 115
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
gtccccagat gagcatgtcc atttttcggc atagcagcgc gcatac 46
<210> 116
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
cagtttttgg ttctacccct ttacaata 28
<210> 117
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
cctccctcac tggagactcg 20
<210> 118
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
ccgacaactt tctcttcagg tcat 24
<210> 119
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
cagcagcttg atggtgccgg 20
<210> 120
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
cacgaaattg attttcaaca tacaac 26
<210> 121
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
gtctttgacc tgaatcttaa caaaatgttg at 32
<210> 122
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
ggtaaacaag tctaccgttc ttatcaac 28
<210> 123
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
catggaccag tttaccagta actgt 25
<210> 124
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
ttgcaacctt ttctgcaaag gtgatttt 28
<210> 125
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
aagaatgaaa gttctgcgtt aacaatgg 28
<210> 126
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
ccaggtgtgt aagggtcacc t 21
<210> 127
<211> 525
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
cggcatagca gcgcgcatac ttttttccac ggccctaggg acaacgtttt tccacggccc 60
tagggacaac gtttttccac ggccctaggg acaacgtttt tccacggccc tagggacaac 120
gtttttccac ggccctaggg acaacgtttt tccacggccc tagggacaac gtttttccac 180
ggccctaggg acaacgtttt tccacggccc tagggacaac gtttttccac ggccctaggg 240
acaacgtttt tccacggccc tagggacaac gtttttccac ggccctaggg acaacgtttt 300
tccacggccc tagggacaac gtttttccac ggccctaggg acaacgtttt tccacggccc 360
tagggacaac gtttttccac ggccctaggg acaacgtttt tccacggccc tagggacaac 420
gtttttccac ggccctaggg acaacgtttt tccacggccc tagggacaac gtttttccac 480
ggccctaggg acaacgtttt tccacggccc tagggacaac gtttt 525
<210> 128
<211> 526
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
cgttgtccct agggccgtgg atttttagtc agcgccgtac caagtgcttt tagtcagcgc 60
cgtaccaagt gcttttagtc agcgccgtac caagtgcttt tagtcagcgc cgtaccaagt 120
gcttttagtc agcgccgtac caagtgcttt tagtcagcgc cgtaccaagt gcttttagtc 180
agcgccgtac caagtgcttt tagtcagcgc cgtaccaagt gcttttagtc agcgccgtac 240
caagtgcttt tagtcagcgc cgtaccaagt gcttttagtc agcgccgtac caagtgcttt 300
tagtcagcgc cgtaccaagt gcttttagtc agcgccgtac caagtgcttt tagtcagcgc 360
cgtaccaagt gcttttagtc agcgccgtac caagtgcttt tagtcagcgc cgtaccaagt 420
gcttttagtc agcgccgtac caagtgcttt tagtcagcgc cgtaccaagt gcttttagtc 480
agcgccgtac caagtgcttt tagtcagcgc cgtaccaagt gctttt 526
<210> 129
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
gcacttggta cggcgctgac t 21
Claims (22)
1. A multiplex nucleic acid detection method comprising the steps of:
1) obtaining a sample containing a target nucleic acid molecule;
2) mixing the sample with at least one set of microspheres pre-coated with target nucleic acid molecule specific probes, and adding at least one set of specific probe combination corresponding to the target nucleic acid molecule, wherein the specific probe combination comprises a capture bridge probe and a label bridge probe;
3) adding at least one group of cascade signal amplification systems, wherein each cascade signal amplification system comprises a primary signal amplification molecule, a secondary signal amplification molecule and a signal probe;
during detection, the target nucleic acid molecule is complementarily hybridized with the pre-coated probe through the capture bridging probe, so as to be immobilized on the microsphere; and the target nucleic acid molecule is connected with the cascade signal amplification system through a labeled bridging probe; the signal probe provides a signal to be detected, the target nucleic acid molecule is detected, and the target nucleic acid in the sample is detected through the detection system.
2. The method of claim 1, wherein the microspheres carry distinguishable labels.
3. The method of claim 2, wherein the distinguishable label is a barcode or a fluorescent dye code.
4. The method of claim 1, wherein the microspheres have a chemically modified and/or magnetic surface.
5. The method of claim 4, wherein the chemical modification of the microsphere surface is a hydroxyl modification.
6. The method of claim 1, wherein the probe combination further comprises a shielding probe for shielding regions of the target nucleic acid molecule from sequences that reduce non-specific hybridization.
7. The method of claim 6, wherein the capture bridge probe, label bridge probe and shielding probe each have a region of complementarity to the target nucleic acid molecule, the region of complementarity being 15-30 nucleotides in sequence length.
8. The method of claim 6, wherein each set of capture bridge probes comprises 2-10 different capture bridge probes having two sequence regions, one sequence region being the target nucleic acid molecule-specific binding region and capable of hybridizing to a specific sequence region of a target nucleic acid molecule, and the other sequence region being a pre-coated probe binding region capable of hybridizing to the pre-coated target nucleic acid molecule-specific probes.
9. The method of claim 6, wherein each set of labeled bridge probes comprises 2-10 different labeled bridge probes, each of the labeled bridge probes having two sequence regions, one sequence region being a binding region specific for the target nucleic acid molecule and capable of hybridizing to a specific sequence region of the target nucleic acid molecule, and the other sequence region being a binding region of the cascade signal amplification system and capable of hybridizing to the cascade signal amplification system.
10. The method of claim 6, wherein each set of shielding probes comprises 2-10 different shielding probes capable of hybridizing to regions of the target nucleic acid molecule that are not hybridized to the capture bridge probes or the label bridge probes.
11. The method of claim 1, wherein said primary signal amplification molecule has two sequence regions, one sequence region being a leader region capable of complementary hybridization to said labeled bridge probe and the other sequence region being comprised of a plurality of repeated sequences capable of hybridization to said primary signal amplification molecule.
12. The method of claim 1, wherein the secondary signal amplification molecule has two sequence regions, one sequence region being a leader region capable of hybridizing to the primary signal amplification molecule and the other sequence region being comprised of a plurality of repeated sequences capable of hybridizing to the signaling probe.
13. The method of claim 1, wherein the signaling probe has two sequence regions, one sequence region being capable of complementary hybridization to a secondary signal amplification molecule and the other sequence region comprising a reporter molecule that directly or indirectly provides a detectable signal or a label that can bind or react with the reporter molecule.
14. The method of claim 13, wherein the detection system is an instrument having a fluorescence reading, a chemiluminescence reading, or a radiation reading capability.
15. The method of claim 14, wherein the detection system comprises a computer and detection analysis software.
16. The method of claim 1, wherein the sample comprises DNA.
17. The method of claim 1, wherein the sample comprises RNA.
18. The method of claim 1, wherein the sample is a compromised sample.
19. The method of claim 18, wherein the compromised sample comprises a paraffin-embedded sample.
20. The method of claim 1, wherein the at least one set of probes specific for the pre-coated target nucleic acid molecule, the at least one set of specific probe combinations, and the at least one set of cascade signal amplification systems are one or more sets.
21. A composition for use in the multiplex nucleic acid detection method of any one of claims 1 to 20, wherein said composition comprises: at least one group of microspheres pre-coated with target nucleic acid specific probes, at least one group of specific probe combinations and at least one group of cascade signal amplification systems; wherein the specific probe combination comprises a capture bridge probe and a label bridge probe; the cascade signal amplification system comprises a primary signal amplification molecule, a secondary signal amplification molecule and a signal probe.
22. A multiplex nucleic acid detection kit, comprising the composition of claim 21.
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Cited By (1)
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| CN114574484A (en) * | 2022-03-17 | 2022-06-03 | 中国科学院北京基因组研究所(国家生物信息中心) | Nucleic acid detection reagent and application thereof |
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