WO2024259563A1 - 一种多重rt-pcr扩增方法及其应用 - Google Patents

一种多重rt-pcr扩增方法及其应用 Download PDF

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Publication number
WO2024259563A1
WO2024259563A1 PCT/CN2023/101078 CN2023101078W WO2024259563A1 WO 2024259563 A1 WO2024259563 A1 WO 2024259563A1 CN 2023101078 W CN2023101078 W CN 2023101078W WO 2024259563 A1 WO2024259563 A1 WO 2024259563A1
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Prior art keywords
sequence
primer
pcr
multiplex
specific
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English (en)
French (fr)
Inventor
张艳艳
杨林
刘锋
杨贵芳
夏军
陈芳
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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Priority to CN202380096088.9A priority Critical patent/CN120917148A/zh
Priority to PCT/CN2023/101078 priority patent/WO2024259563A1/zh
Publication of WO2024259563A1 publication Critical patent/WO2024259563A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the invention belongs to the technical field of multiple RT-PCR amplification, and specifically relates to a multiple RT-PCR amplification method and application thereof.
  • ALK-EML gene fusion can be used for targeted drug therapy of non-small lung cancer
  • micro RNA and ctDNA can be used for early screening and diagnosis of tumors
  • hsp65 sequences can be used for identification and typing of Mycobacterium tuberculosis.
  • RT-qPCR the most commonly used molecular detection technology
  • RT-qPCR can detect fewer targets at a time, its application range is limited.
  • the invention of high-resolution melting curves makes it possible for RT-qPCR to detect multiple targets in one tube, greatly expanding the application range of RT-qPCR.
  • the biggest difficulty is that the RT step and the PCR step are in one reaction.
  • the by-products in the RT process will seriously affect the subsequent PCR amplification, which is mainly manifested in: 1) Specific primers are easy to non-specifically bind to the template during the RT process to produce non-specific products, and exponentially accumulate in the subsequent PCR amplification, that is, in the RT reaction of multi-target RT-qPCR, there will be a local match between the 3 ends of different specific primers and the template, and the RT enzyme will undergo an extension reaction under the condition of local match, resulting in the generation of non-specific products and exponential amplification in the subsequent PCR reaction; 2) Specific primers are easy to bind to each other and extend to form dimers during the RT process, which accumulate exponentially in the subsequent PCR process, resulting in premature termination of the entire reaction, that is, in the RT reaction of multi-target RT-qPCR, there will be a local match between the 3 ends of different specific primers, and the reverse transcriptase will undergo an extension reaction under the condition of local match to produce dimers, which will exponential
  • the first aspect of the present invention aims to provide RT-PCR primers.
  • the second aspect of the present invention aims to provide a kit.
  • the third aspect of the present invention aims to provide a multiplex RT-PCR amplification method.
  • the first aspect of the present invention provides RT-PCR primers, comprising: an upstream primer and a downstream primer;
  • the upstream primer comprises, from the 5' end to the 3' end, a first sequence and a second sequence:
  • the first sequence is a sequence that is completely complementary to the upstream specific sequence
  • the second sequence is completely complementary to the first sequence (i.e., the second sequence is the upstream specific sequence), i.e., the upstream primer presents a completely complementary hairpin structure
  • the first sequence is a sequence that is completely complementary to the upstream specific sequence
  • the second sequence is partially complementary to the first sequence
  • the second sequence is a partial sequence of the upstream specific sequence (preferably a 5' end sequence of the upstream specific sequence)
  • the upstream primer presents a hairpin structure with partial complementarity at the 3' end (i.e., a hairpin structure with a 5' overhang); or
  • the second sequence is an upstream specific sequence
  • the first sequence is partially complementary to the second sequence (i.e. the first sequence is a sequence complementary to a portion of the upstream specific sequence (preferably a sequence complementary to the 5' end sequence of the upstream specific sequence)), that is, the upstream primer presents a hairpin structure with a partially complementary 5' end (i.e. a hairpin structure with a 3' overhang);
  • the downstream primer comprises, from the 5' end to the 3' end, a third sequence and a fourth sequence:
  • the third sequence is a sequence that is completely complementary to the downstream specific sequence
  • the fourth sequence is completely complementary to the third sequence (i.e.
  • the fourth sequence is a downstream specific sequence), that is, the downstream primer presents a completely complementary hairpin structure;
  • the third sequence is a sequence that is completely complementary to the downstream specific sequence
  • the fourth sequence is partially complementary to the third sequence (i.e., the fourth sequence is a partial sequence of the downstream specific sequence (preferably the 5' end sequence of the downstream specific sequence)), that is, the downstream primer presents a hairpin structure with partial complementarity at the 3' end (i.e., a hairpin structure with a 5' overhang); or
  • the fourth sequence is a downstream specific sequence
  • the third sequence is partially complementary to the fourth sequence (i.e. the third sequence is a sequence complementary to a portion of the downstream specific sequence (preferably a sequence complementary to the 5' end sequence of the downstream specific sequence)), that is, the downstream primer presents a hairpin structure with a partially complementary 5' end (i.e. a hairpin structure with a 3' overhang);
  • the 5' end chain of the upstream primer contains one or more cleavage sites that can be cleaved by a specific cleavage agent;
  • the 5' end chain of the downstream primer contains one or more cleavage sites that can be cleaved by a specific cleavage agent.
  • the specific cleavage agent comprises an enzymatic cleavage agent.
  • the enzymatic cleavage agent comprises at least one of a ribonuclease (e.g., ribonuclease H), a glycosylase (e.g., uracil-DNA glycosylase (UDG), a USER enzyme (e.g., a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII), a 3-methyladenine DNA glycosylase, a 3-methyladenine DNA glycosylase II, a pyrimidine hydrate-DNA glycosylase, a FaPy-DNA glycosylase, and a thymine mismatch-DNA glycosylase (e.g., a hypoxanthine-DNA glycosylase, a uracil DNA glycosylase (UDG), a 5-hydroxymethyluracil DNA glycosylase (HmUDG), a 5-hydroxymethylcytosine DNA glycosylase, and a 1,
  • the cleavage site comprises at least one of c1) to c2):
  • rNTP Ribonucleotides
  • the cleavage site comprises c1).
  • the enzymatic cleavage agent comprises at least one of a ribonuclease (e.g., ribonuclease H), a glycosylase (e.g., uracil-DNA glycosylase (UDG), a USER enzyme (e.g., a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII); further, it is a ribonuclease (e.g., ribonuclease H).
  • a ribonuclease e.g., ribonuclease H
  • a glycosylase e.g., uracil-DNA glycosylase (UDG)
  • UDG uracil-DNA glycosylase
  • USER enzyme e.g., a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII
  • the ribonuclease H comprises at least one of ribonuclease HI and ribonuclease HII.
  • the number of cleavage sites that can be cleaved by a specific cleavage agent is an integer.
  • the multiple cleavage sites that can be cleaved by a specific cleavage agent are randomly distributed in the 5' end chain of the upstream primer.
  • the multiple cleavage sites that can be cleaved by a specific cleavage agent are randomly distributed in the 5' end chain of the downstream primer.
  • the first sequence is directly or indirectly linked to the second sequence.
  • the first sequence and the second sequence are connected by any one of the following methods: base connection, linker connection (for example: spacer (spacer3, spacer6, spacer9, spacer18, etc.)).
  • base connection for example: spacer (spacer3, spacer6, spacer9, spacer18, etc.)
  • linker connection for example: spacer (spacer3, spacer6, spacer9, spacer18, etc.)
  • the third sequence is directly or indirectly linked to the fourth sequence.
  • the third sequence and the fourth sequence are connected by any one of the following methods: base connection, linker connection (for example: spacer (spacer3, spacer6, spacer9, spacer18, etc.)).
  • base connection for example: spacer (spacer3, spacer6, spacer9, spacer18, etc.)
  • linker connection for example: spacer (spacer3, spacer6, spacer9, spacer18, etc.)
  • the 5' end strand of the upstream primer comprises a first sequence.
  • the 5' end strand of the downstream primer comprises a third sequence.
  • the first sequence has 20-60 nucleotides.
  • the third sequence has 20 to 60 nucleotides.
  • the second sequence has 5-65 nucleotides.
  • the fourth sequence has 5 to 65 nucleotides.
  • the difference in the number of nucleotides between the first sequence and the second sequence is 1 to 55.
  • the difference in the number of nucleotides between the second sequence and the first sequence is 3 to 10.
  • the difference in the number of nucleotides between the third sequence and the fourth sequence is 1 to 55.
  • the difference in the number of nucleotides between the fourth sequence and the third sequence is 3 to 10.
  • the cleavage site comprises a nucleotide that can be cleaved by a specific cleavage agent.
  • the specific cleavage agents in the upstream primer and the downstream primer may be the same or different.
  • the multiple specific cleavage agents in the upstream primer may be the same or different.
  • the multiple specific cleavage agents in the downstream primer may be the same or different.
  • the cleavage sites in the upstream primer and the downstream primer may be the same or different.
  • the multiple cleavage sites in the upstream primer may be the same or different.
  • the multiple cleavage sites in the downstream primer may be the same or different.
  • the upstream specific sequence and the downstream specific sequence are respectively identical to or complementary to the two segments of the target region.
  • the RT-PCR primers comprise multiple pairs of RT-PCR primers.
  • the second aspect of the present invention provides a kit comprising: the RT-PCR primers of the first aspect of the present invention.
  • the kit further comprises: the specific cleavage agent described in the first aspect of the present invention.
  • the kit further comprises: a DNA polymerase having 3'-5' exonuclease activity (e.g., T4 DNA polymerase, DNA polymerase I (Klenow) large fragment, DNA polymerase I (E. coli)).
  • a DNA polymerase having 3'-5' exonuclease activity e.g., T4 DNA polymerase, DNA polymerase I (Klenow) large fragment, DNA polymerase I (E. coli)
  • a DNA polymerase having 3'-5' exonuclease activity e.g., T4 DNA polymerase, DNA polymerase I (Klenow) large fragment, DNA polymerase I (E. coli)
  • the kit further comprises: reverse transcriptase.
  • the kit further comprises: a reverse transcription primer.
  • the reverse transcription primer is a random primer of 6 to 12 nt; further, a random primer (N6).
  • the reverse transcriptase comprises at least one of AMV reverse transcriptase and M-MuLV reverse transcriptase.
  • the kit comprises: a nucleic acid extraction reagent combination.
  • the nucleic acid extraction reagent combination is specifically a nucleic acid extraction reagent combination for any one of the following methods: alkaline lysis method, phenol chloroform extraction method, chelating resin method, centrifugal column membrane adsorption method and magnetic bead method.
  • the nucleic acid extraction reagent combination comprises: at least one of: a lysis solution, a washing solution, an eluent, and a nucleic acid adsorbent; further preferably, the nucleic acid extraction reagent combination comprises: a lysis solution, a washing solution, an eluent, and a nucleic acid adsorbent.
  • the nucleic acid adsorbent comprises at least one of magnetic beads and adsorption membranes.
  • the kit is used for any one of d1) to d3):
  • the kit when the kit is used to detect a target region, the kit further comprises: at least one of a detection probe and a nucleic acid dye.
  • the detection probe comprises at least one of a TaqMan probe, a molecular beacon, a double hybridization probe, and a composite probe; further It is a TaqMan probe.
  • the nucleic acid dye comprises at least one of EtBr, SYBR Green, SYBR Gold, GelRed, and GelGreen.
  • the third aspect of the present invention provides a multiplex RT-PCR amplification method, comprising the step of using the RT-PCR primers of the first aspect of the present invention.
  • the multiplex RT-PCR amplification method comprises the following steps: performing multiplex RT-PCR reaction on RNA of the sample to be tested using the RT-PCR primers of the first aspect of the present invention.
  • reaction further comprises:
  • the multiplex RT-PCR reaction system is treated with the specific cleavage agent.
  • the reverse transcription (RT) reaction and amplification (PCR) reaction of the multiplex RT-PCR reaction are performed in the same system.
  • the same system is specifically carried out in the same reaction system; that is, there is no need to first carry out a reverse transcription reaction in one reaction system and then carry out an amplification reaction in another reaction system.
  • the multiplex RT-PCR reaction further comprises a step of extending the upstream primer and/or the downstream primer to obtain an upstream primer and/or a downstream primer with a completely complementary hairpin structure.
  • the step of extending the upstream primer and/or the downstream primer is performed simultaneously with the RT reaction of the multiplex RT-PCR reaction.
  • the method of treating the multiplex RT-PCR reaction system with the specific cleavage agent may be:
  • the multiplex RT-PCR reaction system comprises the specific cleavage agent.
  • the step of treating the multiplex RT-PCR reaction system with the specific cleavage agent is performed during the RT reaction of the multiplex RT-PCR reaction or after the RT reaction of the multiplex RT-PCR reaction.
  • the multiplex RT-PCR reaction system further comprises: a DNA polymerase having 3'-5' exonuclease activity (preferably the DNA polymerase having 3'-5' exonuclease activity in the second aspect of the present invention).
  • a DNA polymerase having 3'-5' exonuclease activity preferably the DNA polymerase having 3'-5' exonuclease activity in the second aspect of the present invention.
  • the multiplex RT-PCR reaction system further comprises: reverse transcriptase.
  • the multiplex RT-PCR reaction system further comprises: reverse transcription primers.
  • the RNA is obtained by a cleavage reaction of the sample to be tested.
  • the cleavage reaction precedes the multiplex RT-PCR reaction.
  • the cleavage reaction and the multiplex RT-PCR reaction are performed in the same system.
  • the same system is specifically the same reaction container.
  • the extraction and purification step is not included after the cleavage reaction and before the multiplex RT-PCR reaction.
  • an extraction and purification step is further included.
  • the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid.
  • the extraction and purification further comprises: eluting or dissolving nucleic acids.
  • the cleavage reaction and the multiplex RT-PCR reaction are performed in different systems, that is, not in the same system.
  • the same system is specifically the same reaction container.
  • the cleavage reaction further comprises: an extraction and purification step.
  • the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid.
  • the extraction and purification further comprises: eluting or dissolving nucleic acids.
  • the lysis method comprises at least one of a physical method, a chemical method and a biological method.
  • the multiplex RT-PCR reaction system further comprises at least one of a detection probe and a nucleic acid dye.
  • the detection probe comprises at least one of a TaqMan probe, a molecular beacon, a dual hybridization probe, and a composite probe;
  • the nucleic acid dye comprises at least one of EtBr, SYBR Green, SYBR Gold, GelRed, and GelGreen.
  • the present invention provides RT-PCR primers.
  • the inventor creatively designs the primers (upstream primers and/or downstream primers) into fully complementary or partially complementary hairpin structures (hairpin structures with 3'-end partial complementarity or hairpin structures with 5'-end partial complementarity), so that the 3'-end of the primer loses its binding ability (no non-specific binding with the template and/or avoids binding between primers) and extension ability during the RT process, and the 5'-end chain of the primer contains one or more cleavage sites that can be cleaved by a specific cleavage agent, so that the cleavage site of the 5'-end chain of the primer is cleaved by the specific cleavage agent during the RT process of RT-PCR (the 3'-end partial complementarity hairpin structure also contains the primer in the reverse transcriptase and/or DNA A step of extending the 5'-terminal chain under the action of a polymerase to obtain a completely complementary hairpin structure), but maintaining
  • FIG. 1 is a schematic diagram of the structure of the primer of the present invention.
  • FIG. 2 is a schematic diagram showing the principle of RT-PCR amplification using primers of the present invention.
  • FIG. 3 is a graph showing the fluorescence detection results of Example 1 and Comparative Example 1.
  • FIG. 4 is an electrophoretic diagram of the amplified products of Example 1 and Comparative Example 1.
  • first”, “second”, “third”, and “fourth” are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features.
  • a feature defined as “first”, “second”, and “third” may explicitly or implicitly include at least one of the features.
  • the meaning of “plurality” is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
  • the first aspect of the present invention provides RT-PCR primers, comprising: an upstream primer and a downstream primer;
  • the upstream primer comprises, from the 5' end to the 3' end, a first sequence and a second sequence:
  • the first sequence is a sequence that is completely complementary to the upstream specific sequence
  • the second sequence is completely complementary to the first sequence (i.e., the second sequence is the upstream specific sequence), i.e., the upstream primer presents a completely complementary hairpin structure
  • the first sequence is a sequence that is completely complementary to the upstream specific sequence
  • the second sequence is partially complementary to the first sequence
  • the second sequence is a partial sequence of the upstream specific sequence (preferably a 5' end sequence of the upstream specific sequence)
  • the upstream primer presents a hairpin structure with partial complementarity at the 3' end (i.e., a hairpin structure with a 5' overhang); or
  • the second sequence is an upstream specific sequence
  • the first sequence is partially complementary to the second sequence (i.e. the first sequence is a sequence complementary to a portion of the upstream specific sequence (preferably a sequence complementary to the 5' end sequence of the upstream specific sequence)), that is, the upstream primer presents a hairpin structure with a partially complementary 5' end (i.e. a hairpin structure with a 3' overhang);
  • the downstream primer comprises, from the 5' end to the 3' end, a third sequence and a fourth sequence:
  • the third sequence is a sequence that is completely complementary to the downstream specific sequence
  • the fourth sequence is completely complementary to the third sequence (i.e., the fourth sequence is the downstream specific sequence), i.e., the downstream primer presents a completely complementary hairpin structure;
  • the third sequence is a sequence that is completely complementary to the downstream specific sequence
  • the fourth sequence is partially complementary to the third sequence (i.e., the fourth sequence is a partial sequence of the downstream specific sequence (preferably the 5' end sequence of the downstream specific sequence)), that is, the downstream primer presents a hairpin structure with partial complementarity at the 3' end (i.e., a hairpin structure with a 5' overhang); or
  • the fourth sequence is a downstream specific sequence
  • the third sequence is partially complementary to the fourth sequence (i.e. the third sequence is a sequence complementary to a portion of the downstream specific sequence (preferably a sequence complementary to the 5' end sequence of the downstream specific sequence)), that is, the downstream primer presents a hairpin structure with a partially complementary 5' end (i.e. a hairpin structure with a 3' overhang);
  • the 5' end chain of the upstream primer contains one or more cleavage sites that can be cleaved by a specific cleavage agent;
  • the 5' end strand of the downstream primer contains one or more cleavage sites that can be cleaved by a specific cleavage agent.
  • the first sequence is a sequence that is completely complementary to the upstream specific sequence
  • the second sequence is completely complementary to the first sequence (i.e., the second sequence is the upstream specific sequence)
  • the third sequence is a sequence that is completely complementary to the downstream specific sequence
  • the fourth sequence is completely complementary to the third sequence (i.e., the fourth sequence is the downstream specific sequence).
  • the inventor creatively designs the primers (upstream primers and/or downstream primers) into fully complementary or partially complementary hairpin structures (partially complementary hairpin structures at the 3' end or partially complementary hairpin structures at the 5' end), so that the 3' end of the primer loses its binding ability (does not non-specifically bind to the template and/or avoids binding between primers) and extension ability during the RT process, and the 5' end chain of the primer contains one or more cleavage sites that can be cleaved by a specific cleavage agent ( Figure 1), so that the cleavage site of the 5' end chain of the primer is
  • the specific cutting agent cuts (for the primers with partially complementary hairpin structures at the 3' end, it also includes the step of extending the primers under the action of reverse transcriptase and/or DNA polymerase to obtain primers with completely complementary hairpin structures), but the complementary double-stranded state is maintained during the RT process; in the subsequent high-temperature process of PCR (such as
  • the primer structure is a completely complementary hairpin structure: a specific sequence (an upstream specific sequence and/or a downstream specific sequence, corresponding to the second sequence and the fourth sequence mentioned above) and an oligonucleotide chain that is completely complementary thereto (e.g., a DNA chain, corresponding to the first sequence and the third sequence mentioned above) form a "closed" hairpin structure, which can prevent the primer from non-specifically binding to the template during the RT process and binding between primers, wherein the 5' end chain of the primer contains one or more cleavage sites that can be cleaved by a specific cleavage agent, so that the cleavage site of the 5' end chain of the primer is cleaved by the specific cleavage agent during the RT process of RT-PCR, but the complementary double-stranded state is maintained during the RT process; in the subsequent high temperature process of PCR (e.g., thermal denaturation), the cleaved 5' end chain is freed,
  • the primer structure is a partially complementary hairpin structure:
  • the primer structure is a hairpin structure with partial complementarity at the 3' end
  • the oligonucleotide chain complementary to the specific sequence (upstream specific sequence and/or downstream specific sequence) (corresponding to the above-mentioned first sequence and third sequence) and the partial specific sequence (the 5' end sequence of the specific sequence, corresponding to the above-mentioned second sequence and fourth sequence) form a hairpin structure with partial complementarity at the 3' end (i.e., a hairpin structure with a 5' protruding end), wherein the 5' end chain of the primer contains one or more cleavage sites that can be cleaved by a specific cleavage agent;
  • the primer is extended under the action of a reverse transcriptase and/or a DNA polymerase to obtain a primer containing the full sequence of the specific sequence and form a completely complementary hairpin structure; and the cleavage site of the 5' end chain of the primer is cleaved by the
  • the specific sequence upstream specific sequence and/or downstream specific sequence, corresponding to the above-mentioned second sequence and fourth sequence
  • the oligonucleotide chain complementary to its partial sequence preferably a sequence complementary to the 5' end sequence of the specific sequence, such as a DNA chain, corresponding to the above-mentioned first sequence and third sequence
  • the 3' end of the primer protruding more than 3 bases can prevent non-specific addition of bases by the reverse transcriptase or can be cut into a blunt end by a DNA polymerase with 3'-5' nuclease activity
  • the 5' end chain of the primer contains one or more cleavage sites that can be cut by a specific cleavage agent, so that the cleavage site of
  • the RT-PCR primers of the present invention can realize one-step amplification and effectively avoid the multiple pairs of primers in the reverse transcriptase during the RT process.
  • dimers are formed, and multiple pairs of primers are non-specifically bound to the template under the action of the reverse transcriptase system to produce non-specific products, which greatly improves the efficiency of PCR amplification and the sensitivity and specificity of detection.
  • the upstream specific sequence and the downstream specific sequence are respectively identical to or complementary to the two segments of the target region.
  • the design of the specific sequences (upstream specific sequences and/or downstream specific sequences) in the primers (upstream primers and/or downstream primers) follows conventional primer design principles, for example: the upstream specific sequence is complementary to the upstream negative chain (i.e., antisense chain, cDNA second chain) sequence of the target region or is identical to the positive chain (i.e., sense chain, cDNA first chain) sequence, and the downstream specific sequence is complementary to the downstream positive chain (i.e., sense chain, cDNA first chain) sequence of the target region or is identical to the negative chain (i.e., antisense chain, cDNA second chain) sequence.
  • the upstream specific sequence is complementary to the upstream negative chain (i.e., antisense chain, cDNA second chain) sequence of the target region or is identical to the positive chain (i.e., sense chain, cDNA first chain) sequence
  • the downstream specific sequence is complementary to the downstream positive chain (i.e., sense chain, cDNA first chain) sequence of the
  • the upstream specific sequence is complementary to the upstream negative strand sequence of the target region or is identical to the positive strand sequence.
  • the downstream specific sequence is complementary to the downstream positive strand sequence of the target region or is identical to the negative strand sequence.
  • the specific cleavage agent comprises an enzymatic cleavage agent.
  • the enzymatic cleavage agent comprises at least one of a ribonuclease (e.g., ribonuclease H), a glycosylase (e.g., uracil-DNA glycosylase (UDG), a USER enzyme (e.g., a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII), a 3-methyladenine DNA glycosylase, a 3-methyladenine DNA glycosylase II, a pyrimidine hydrate-DNA glycosylase, a FaPy-DNA glycosylase, and a thymine mismatch-DNA glycosylase (e.g., a hypoxanthine-DNA glycosylase, a uracil DNA glycosylase (UDG), a 5-hydroxymethyluracil DNA glycosylase (HmUDG), a 5-hydroxymethylcytosine DNA glycosylase, and a 1,
  • the cleavage site comprises at least one of c1) to c2):
  • rNTPs ribonucleotides
  • rATP ribonucleotide
  • rGTP ribonucleotide
  • tCTP ribonuclease
  • rUTP ribonuclease
  • its corresponding specific cleavage agent may also be uracil-DNA glycosylase (UDG) or USER enzyme (a mixture of uracil-DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII);
  • dUTP uracil deoxyribonucleotide
  • UDG uracil-DNA glycosylase
  • USER enzyme a mixture of uracil-DNA glycosylase (UDG) and DNA glycosylase-cleavage enzyme Endo VIII
  • the cleavage site comprises c1).
  • the enzymatic cleavage agent comprises at least one of a ribonuclease (e.g., ribonuclease H), a glycosylase (e.g., uracil-DNA glycosylase (UDG), a USER enzyme (e.g., a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII); further, it is a ribonuclease (e.g., ribonuclease H).
  • a ribonuclease e.g., ribonuclease H
  • a glycosylase e.g., uracil-DNA glycosylase (UDG)
  • UDG uracil-DNA glycosylase
  • USER enzyme e.g., a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII
  • the ribonuclease H comprises at least one of ribonuclease HI and ribonuclease HII.
  • the specific cleavage agents in the upstream primer and the downstream primer may be the same or different.
  • the multiple specific cleavage agents in the upstream primer may be the same or different.
  • the multiple specific cleavage agents in the downstream primer may be the same or different.
  • the cleavage sites in the upstream primer and the downstream primer may be the same or different.
  • the multiple cleavage sites in the upstream primer may be the same or different.
  • the multiple cleavage sites in the downstream primer may be the same or different.
  • the number of cleavage sites that can be cleaved by a specific cleavage agent is an integer.
  • the plurality of cleavage sites that can be cleaved by a specific cleavage agent are randomly distributed in the 5' end chain of the upstream primer.
  • the multiple cleavage sites that can be cleaved by a specific cleavage agent are randomly distributed in the 5' end chain of the downstream primer.
  • the first sequence is directly or indirectly linked to the second sequence.
  • the first sequence and the second sequence are connected by any one of the following methods: base connection, linker connection (for example: spacer (spacer3, spacer6, spacer9, spacer18, etc.)).
  • base connection for example: spacer (spacer3, spacer6, spacer9, spacer18, etc.)
  • linker connection for example: spacer (spacer3, spacer6, spacer9, spacer18, etc.)
  • the third sequence is directly or indirectly linked to the fourth sequence.
  • the third sequence and the fourth sequence are connected by any one of the following methods: base connection, linker connection (for example: spacer (spacer3, spacer6, spacer9, spacer18, etc.)).
  • base connection for example: spacer (spacer3, spacer6, spacer9, spacer18, etc.)
  • linker connection for example: spacer (spacer3, spacer6, spacer9, spacer18, etc.)
  • the 5' end strand of the upstream primer comprises a first sequence.
  • the 5' end chain of the downstream primer contains a third sequence.
  • the first sequence has 20-60 nucleotides.
  • the third sequence has 20 to 60 nucleotides.
  • the second sequence has 5-65 nucleotides.
  • the fourth sequence has 5 to 65 nucleotides.
  • the difference in the number of nucleotides between the first sequence and the second sequence is 1 to 55.
  • the difference in the number of nucleotides between the second sequence and the first sequence is 3 to 10.
  • the difference in the number of nucleotides between the third sequence and the fourth sequence is 1 to 55.
  • the difference in the number of nucleotides between the fourth sequence and the third sequence is 3 to 10.
  • the cleavage site comprises a nucleotide that can be cleaved by a specific cleavage agent.
  • the RT-PCR primer is a DNA fragment or an analog thereof.
  • the RT-PCR primers comprise multiple pairs of RT-PCR primers.
  • the plurality of pairs are two or more pairs.
  • the RT-PCR primers for detecting the SARS-COV-2 gene include: an upstream primer and a downstream primer;
  • sequences of the upstream primers are shown in SEQ ID NOs. 1, 4, 7, 10, and 13, and the sequences of the downstream primers are shown in SEQ ID NOs. 2, 5, 8, 11, and 14;
  • the 5' ends of the upstream primers with sequences such as SEQ ID NO. 1, 4, 7, 10, and 13 each independently contain one or more cleavage sites that can be cleaved by a specific cleavage agent;
  • the 5’ end of the downstream primers with sequences such as SEQ ID NO. 2, 5, 8, 11, and 14 independently contains one or more cleavage sites that can be cleaved by a specific cleavage agent.
  • the specific cleavage agent comprises an enzymatic cleavage agent.
  • the enzymatic cleavage agent comprises at least one of a ribonuclease (e.g., ribonuclease H), a glycosylase (e.g., uracil-DNA glycosylase (UDG), a USER enzyme (e.g., a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII), a 3-methyladenine DNA glycosylase, a 3-methyladenine DNA glycosylase II, a pyrimidine hydrate-DNA glycosylase, a FaPy-DNA glycosylase, and a thymine mismatch-DNA glycosylase (e.g., a hypoxanthine-DNA glycosylase, a uracil DNA glycosylase (UDG), a 5-hydroxymethyluracil DNA glycosylase (HmUDG), a 5-hydroxymethylcytosine DNA glycosylase, and a 1,
  • the cleavage site comprises at least one of c1) to c2):
  • ribonucleotides for example: rATP, rGTP, tCTP, rUTP
  • the corresponding specific cutting agent is ribonuclease (for example For example, ribonuclease H)
  • the corresponding specific cleavage agent may also be uracil-DNA glycosylase (UDG) or USER enzyme (for example, a mixture of uracil-DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII);
  • dUTP uracil deoxyribonucleotide
  • UDG uracil-DNA glycosylase
  • USER enzyme for example: a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-cleavage enzyme Endo VIII.
  • the cleavage site comprises c1).
  • the enzymatic cleavage agent comprises at least one of a ribonuclease (e.g., ribonuclease H), a glycosylase (e.g., uracil-DNA glycosylase (UDG), a USER enzyme (e.g., a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII); further, a ribonuclease (e.g., ribonuclease H).
  • a ribonuclease e.g., ribonuclease H
  • a glycosylase e.g., uracil-DNA glycosylase (UDG)
  • UDG uracil-DNA glycosylase
  • USER enzyme e.g., a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII
  • the ribonuclease H comprises at least one of ribonuclease HI and ribonuclease HII.
  • the positions of the ribonucleotides of the upstream primers having sequences as shown in SEQ ID NO.1, 4, 7, 10, 13 are as shown in Table 1 and notes.
  • the positions of the ribonucleotides of the downstream primers whose sequences are shown in SEQ ID NO. 2, 5, 8, 11, 14 are as shown in Table 2 and notes.
  • the RT-PCR primers are used to amplify RNA of the sample to be tested.
  • the sample to be tested comprises cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, and nasal swabs.
  • the body fluid comprises at least one of tissue fluid, lymph fluid, blood and cerebrospinal fluid.
  • the microorganism comprises at least one of bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, and spirochetes.
  • the RNA comprises at least one of mRNA, lncRNA, small RNA, cfRNA and tRNA.
  • the small RNA may be at least one of miRNA, siRNA and piRNA.
  • the second aspect of the present invention provides a kit comprising: the RT-PCR primers of the first aspect of the present invention.
  • the kit further comprises: the specific cutting agent, which is used to cut the cleavage site of the 5' end chain of the primer (upstream primer and/or downstream primer) of the hairpin structure that is completely complementary or partially complementary during the RT process of RT-PCR.
  • the specific cutting agent which is used to cut the cleavage site of the 5' end chain of the primer (upstream primer and/or downstream primer) of the hairpin structure that is completely complementary or partially complementary during the RT process of RT-PCR.
  • the kit further comprises: a DNA polymerase with 3'-5' exonuclease activity (for example: T4 DNA polymerase, DNA polymerase I (Klenow) large fragment, DNA polymerase I (E. coli)), which is used to maintain the 3' end blunt end structure of the primer (upstream primer and/or downstream primer) (inhibiting the terminal transfer activity of reverse transcriptase), and the DNA polymerase can also be used for primer (upstream primer and/or downstream primer) extension to obtain a primer containing the full sequence of the specific sequence and form a completely complementary hairpin structure (when the second sequence is partially complementary to the first sequence and/or the fourth sequence is partially complementary to the third sequence).
  • a DNA polymerase with 3'-5' exonuclease activity for example: T4 DNA polymerase, DNA polymerase I (Klenow) large fragment, DNA polymerase I (E. coli)
  • the DNA polymerase can also be used for primer (upstream primer and/or downstream primer) extension to obtain
  • the kit further comprises: a reverse transcription reaction mixture, which is used for reverse transcription reaction (ie, reverse transcribing RNA into cDNA).
  • a reverse transcription reaction mixture which is used for reverse transcription reaction (ie, reverse transcribing RNA into cDNA).
  • the reverse transcription reaction mixture comprises: at least one of reverse transcriptase, DNA polymerase, reverse transcription buffer, reverse transcription primer, dNTPs, and RNase inhibitor; preferably, the reverse transcription reaction mixture comprises: reverse transcriptase, DNA polymerase, reverse transcription buffer, dNTPs, RNase inhibitor and reverse transcription primer; the reverse transcriptase and/or DNA polymerase can also be used for primer (upstream primer and/or downstream primer) extension to obtain primers containing the full sequence of the specific sequence and form a completely complementary hairpin structure (when the primer structure is a hairpin structure with partial complementarity at the 3' end); for the components already contained in the kit, the reverse transcription reaction mixture can reduce the components accordingly: for example: when the kit contains a DNA polymerase with 3'-5' nuclease activity, the reverse transcription reaction mixture may not contain DNA polymerase.
  • the reverse transcription primer is a random primer of 6 to 12 nt; further, a random primer (N6).
  • the reverse transcriptase comprises at least one of AMV reverse transcriptase and M-MuLV reverse transcriptase.
  • the kit further comprises: a PCR reaction mixture, which is used for PCR reaction.
  • the PCR reaction mixture comprises: at least one of: DNA polymerase, Mg 2+ , PCR buffer, and dNTPs; further preferably, the PCR reaction mixture further comprises: DNA polymerase, Mg 2+ , PCR buffer, and dNTPs; wherein the DNA polymerase
  • the enzyme can also be used to extend primers (upstream primers and/or downstream primers) to obtain primers containing the entire sequence of the specific sequence and to form a completely complementary hairpin structure (when the primer structure is a hairpin structure with partial complementarity at the 3'end); for components already contained in the kit, the PCR reaction mixture can reduce the components accordingly, for example: when the kit contains a DNA polymerase with 3'-5' nuclease activity, and/or the reverse transcription reaction solution of the kit contains a DNA polymerase, the PCR reaction mixture may not contain a DNA polymerase.
  • the DNA polymerase is a thermostable DNA polymerase (e.g., Taq DNA polymerase); further, it is a Taq hot-start DNA polymerase.
  • a thermostable DNA polymerase e.g., Taq DNA polymerase
  • Taq hot-start DNA polymerase e.g., Taq hot-start DNA polymerase
  • the kit comprises: a nucleic acid extraction reagent combination.
  • the nucleic acid extraction reagent combination is specifically a nucleic acid extraction reagent combination for any one of the following methods: alkaline lysis method, phenol chloroform extraction method, chelating resin method, centrifugal column membrane adsorption method and magnetic bead method.
  • the nucleic acid extraction reagent combination comprises: at least one of: a lysis solution, a washing solution, an eluent, and a nucleic acid adsorbent; further preferably, the nucleic acid extraction reagent combination comprises: a lysis solution, a washing solution, an eluent, and a nucleic acid adsorbent.
  • the nucleic acid adsorbent comprises at least one of magnetic beads and adsorption membranes.
  • the kit is used for any one of d1) to d3):
  • the kit when the kit is used to detect a target area, the kit also comprises: at least one of a detection probe and a nucleic acid dye; and further comprises a detection probe, which is a probe commonly used in existing RT-qPCR and can be designed by a person skilled in the art based on the target area to be detected.
  • a detection probe which is a probe commonly used in existing RT-qPCR and can be designed by a person skilled in the art based on the target area to be detected.
  • the detection probe comprises at least one of a TaqMan probe, a molecular beacon, a dual hybridization probe, and a composite probe; further, the detection probe is a TaqMan probe.
  • the nucleic acid dye comprises at least one of EtBr, SYBR Green, SYBR Gold, GelRed, and GelGreen.
  • the RT-PCR primers are the RT-PCR primers for detecting the SARS-COV-2 gene of the first aspect of the present invention.
  • the kit when the kit is used to detect the SARS-COV-2 gene, the kit further comprises a probe for detecting the SARS-COV-2 gene, and the sequence of the probe for detecting the SARS-COV-2 gene is shown in SEQ ID NO. 3, 6, 9, 10, 15.
  • the two ends of the probe for detecting the SARS-COV-2 gene are respectively connected to a quenching group and a fluorescent group.
  • the fluorescent group includes at least one of FAM, TET, VIC, HEX, Cy5, ROX, Texas Red, Tamara, and JOE; further preferably, the fluorescent group includes FAM.
  • the quencher group comprises at least one of BHQ1, BHQ2, BHQ3, and MGB; further preferably, the quencher group comprises BHQ1.
  • the kit when the kit is used to construct a library of a target region, the kit further comprises: a reagent for adding a sequencing adapter.
  • the reagent for adding sequencing adapters is selected from any one of the following methods for adding sequencing adapters: a method for adding sequencing adapters by PCR, a method for adding sequencing adapters by ligase.
  • the third aspect of the present invention provides a sequencing reagent kit, comprising: a reagent kit using the second aspect of the present invention.
  • the sequencing reagent set further comprises: a sequencing reagent kit.
  • a fourth aspect of the present invention provides a sequencing system, comprising: a sequencer and the sequencing reagent set of the third aspect of the present invention.
  • the fifth aspect of the present invention provides a multiplex RT-PCR amplification method, comprising the step of using the RT-PCR primers of the first aspect of the present invention.
  • the multiplex RT-PCR amplification method comprises the following steps: performing multiplex RT-PCR reaction on RNA of the sample to be tested using the RT-PCR primers of the first aspect of the present invention.
  • reaction further comprises:
  • the multiplex RT-PCR reaction system is treated with the specific cleavage agent.
  • the reverse transcription (RT) reaction and amplification (PCR) reaction of the multiplex RT-PCR reaction are performed in the same system.
  • the same system is specifically carried out in the same reaction system; that is, there is no need to first carry out a reverse transcription reaction in one reaction system and then carry out an amplification reaction in another reaction system.
  • the multiplex RT-PCR reaction further comprises a step of extending the upstream primer and/or the downstream primer to obtain an upstream primer and/or a downstream primer with a completely complementary hairpin structure.
  • the step of extending the upstream primer and/or the downstream primer is performed simultaneously with the RT reaction of the multiplex RT-PCR reaction.
  • the primers are designed to be completely complementary or partially complementary hairpin structures (hairpin structures with partial complementarity at the 3' end or hairpin structures with partial complementarity at the 5' end), so that the 3' end of the primer loses the ability to bind (does not bind to the template non-specifically and/or avoids binding between primers) and the ability to extend during the RT process, and the 5' end chain of the primer contains one or more cleavage sites that can be cleaved by a specific cleavage agent, so that the cleavage site of the 5' end chain of the primer is cleaved by a specific cleavage agent during the RT process of RT-PCR (the 3' end partially complementary hairpin structure also contains the primer in the reverse transcriptase and/or DNA polymerase
  • the method comprises the following steps: a step of extending the 5' end chain under the action of the reverse transcriptase system to obtain a
  • the step of treating the multiplex RT-PCR reaction system with the specific cleavage agent is performed during the RT reaction of the multiplex RT-PCR reaction or after the RT reaction of the multiplex RT-PCR reaction.
  • the method of treating the multiplex RT-PCR reaction system with the specific cleavage agent may be:
  • the multiplex RT-PCR reaction system comprises the specific cleavage agent.
  • the system of the multiplex RT-PCR reaction further comprises: a DNA polymerase with 3’-5’ exonuclease activity (preferably the DNA polymerase with 3’-5’ exonuclease activity in the second aspect of the present invention), which is used to maintain the blunt end structure of the 3’ end of the primer (upstream primer and/or downstream primer) (inhibiting the terminal transfer activity of reverse transcriptase), and the DNA polymerase can also be used to extend the primer (upstream primer and/or downstream primer) to obtain a primer containing the full sequence of the specific sequence and form a completely complementary hairpin structure (when the primer structure is a partially complementary hairpin structure at the 3’ end).
  • a DNA polymerase with 3’-5’ exonuclease activity preferably the DNA polymerase with 3’-5’ exonuclease activity in the second aspect of the present invention
  • the DNA polymerase can also be used to extend the primer (upstream primer and/or downstream primer) to obtain a primer containing the full sequence
  • the multiplex RT-PCR reaction system further comprises: a reverse transcription reaction mixture, which is used for reverse transcription reaction (ie, reverse transcribing RNA into cDNA).
  • a reverse transcription reaction mixture which is used for reverse transcription reaction (ie, reverse transcribing RNA into cDNA).
  • the reverse transcription reaction mixture contains: at least one of reverse transcriptase, DNA polymerase, reverse transcription buffer, reverse transcription primer, dNTPs, and RNase inhibitor; preferably, the reverse transcription reaction mixture contains: reverse transcriptase, DNA polymerase, reverse transcription buffer, dNTPs, RNase inhibitor and reverse transcription primer; the reverse transcriptase and/or DNA polymerase can also be used for primer (upstream primer and/or downstream primer) extension to obtain primers containing the full sequence of the specific sequence and form a completely complementary hairpin structure (when the primer structure is a hairpin structure with partial complementarity at the 3' end); for the components already contained in the RT-PCR reaction system, the reverse transcription reaction mixture can reduce the components accordingly: for example: when the RT-PCR reaction system contains a DNA polymerase with 3'-5' nuclease activity, the reverse transcription reaction mixture may not contain DNA polymerase.
  • the reverse transcription primer is a random primer of 6 to 12 nt; further, a random primer (N6).
  • the reverse transcriptase comprises at least one of AMV reverse transcriptase and M-MuLV reverse transcriptase.
  • the multiplex RT-PCR reaction system further comprises: a PCR reaction mixture, which is used for PCR reaction.
  • the PCR reaction mixture comprises: at least one of DNA polymerase, Mg 2+ , PCR buffer, and dNTPs;
  • the PCR reaction mixture further comprises: DNA polymerase, Mg2 + , PCR buffer and dNTPs; the DNA polymerase can also be used for primer (upstream primer and/or downstream primer) extension to obtain primers containing the full sequence of the specific sequence and form a completely complementary hairpin structure (when the primer structure is a hairpin structure with partial complementarity at the 3'end); for components already contained in the RT-PCR reaction system, the PCR reaction mixture can reduce the components accordingly, for example: when the RT-PCR reaction system contains a DNA polymerase with 3'-5' nuclease activity, and/or the reverse transcription reaction solution of the RT-PCR reaction system contains a DNA polymerase, the PCR reaction mixture may not contain a DNA polymerase.
  • the DNA polymerase is a thermostable DNA polymerase (e.g., Taq DNA polymerase); further, it is a Taq hot-start DNA polymerase.
  • a thermostable DNA polymerase e.g., Taq DNA polymerase
  • Taq hot-start DNA polymerase e.g., Taq hot-start DNA polymerase
  • the concentration of the specific cleavage agent in the multiplex RT-PCR reaction system is lower than the concentration of the enzyme used for extension (eg, the DNA polymerase and/or the reverse transcriptase), so that the extension reaction precedes the cleavage reaction.
  • the enzyme used for extension eg, the DNA polymerase and/or the reverse transcriptase
  • the RNA is obtained by a cleavage reaction of the sample to be tested.
  • the cleavage reaction precedes the multiplex RT-PCR reaction.
  • the cleavage reaction and the multiplex RT-PCR reaction are performed in the same system.
  • the same system is specifically the same reaction container.
  • the extraction and purification step is not included after the cleavage reaction and before the multiplex RT-PCR reaction.
  • an extraction and purification step is further included to remove impurities such as salts and organic agents.
  • the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid.
  • the extraction and purification further comprises: eluting or dissolving nucleic acids.
  • the cleavage reaction and the multiplex RT-PCR reaction are performed in different systems, that is, not in the same system.
  • the same system is specifically the same reaction container.
  • the cleavage reaction further comprises: an extraction and purification step to remove impurities such as salts and organic agents.
  • the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid.
  • the extraction and purification further comprises: eluting or dissolving nucleic acids.
  • the lysis method comprises at least one of a physical method, a chemical method and a biological method.
  • the physical method comprises: at least one of a boiling method, a glass bead method, an ultrasonic method, a grinding method, a freeze-thaw method, and a homogenization method.
  • the chemical method comprises at least one of a surfactant method (SDS method) and an alkaline lysis method.
  • SDS method surfactant method
  • alkaline lysis method alkaline lysis method
  • the biological method comprises: an enzymatic method, such as cleavage by enzymes such as lysozyme and proteinase K.
  • the sixth aspect of the present invention provides a method for detecting a target region, comprising the steps of the multiplex RT-PCR amplification method of the fifth aspect of the present invention.
  • the multiplex RT-PCR reaction system further comprises a detection probe and/or a nucleic acid dye (preferably the detection probe and/or nucleic acid dye in the second aspect of the present invention).
  • a method for detecting SARS-COV-2 gene comprises the following steps: performing multiple RT-PCR reactions on RNA of a sample to be tested using the RT-PCR primers for detecting SARS-COV-2 gene in the first aspect of the present invention.
  • the RT-PCR reaction system also includes the probe for detecting the SARS-COV-2 gene in the second aspect of the present invention.
  • the method is a method for non-diagnostic purposes.
  • the seventh aspect of the present invention provides a method for constructing a targeted library, comprising the steps of the multiplex RT-PCR amplification method of the fifth aspect of the present invention
  • the method further comprises the following steps: adding a sequencing adapter to the RT-PCR product to obtain a sequencing library.
  • a circularization reaction may be further included after adding the sequencing adapter before obtaining the sequencing library, that is, circularizing the linear library obtained by amplification.
  • the method further comprises the step of: purifying the sequencing library.
  • the purification is performed using magnetic beads.
  • the eighth aspect of the present invention provides a sequencing method, comprising: the steps of the method for constructing a targeted library according to the seventh aspect of the present invention.
  • the sequencing method comprises the following steps: preparing a library; sequencing;
  • the method for preparing a library is the method for constructing a targeted library according to the seventh aspect of the present invention.
  • the sequencing further includes the following steps: library quality inspection.
  • the ninth aspect of the present invention provides a method for obtaining gene information of a target region, comprising: the steps of the sequencing method of the eighth aspect of the present invention.
  • the method for obtaining the target region gene information comprises the following steps: sequencing to obtain sequencing data; obtaining the target region gene information; the information comprises at least one of sequence information and variation information;
  • the sequencing method is the sequencing method of the eighth aspect of the present invention.
  • the method for obtaining the target region gene information further comprises the following steps: comparing the obtained sequencing data with the reference genome to determine the variation information of the target region gene.
  • the variation includes at least one of gene fusion, SNV, gene deletion, and gene insertion mutation.
  • the method is a method for non-diagnostic purposes.
  • the present invention provides use of the RT-PCR primers of the first aspect, the kit of the second aspect, the sequencing reagent set of the third aspect and/or the sequencing system of the fourth aspect in any one of items f1) to f10);
  • the applications described in f5) and f9) are for non-diagnostic purposes.
  • the method of the following embodiment/comparative example is used to detect multiple gene sequences of the new coronavirus, including 5 pairs of primers, which respectively amplify 5 target sequences to increase the probability of detecting the new coronavirus-specific sequence;
  • the RT-qPCR reagent is from Novozymes (HiScript II One Step RT-PCR Kit, product number P611-01), the RNase HII enzyme is from NEB, product number M0288S, and the Klenow enzyme is from NEB, USA, product number M0210S.
  • Example 1 A method for detecting SARS-COV-2 genes using multiplex RT-qPCR
  • a method for detecting SARS-COV-2 genes by multiplex RT-qPCR comprising the following steps:
  • the RT-qPCR system was configured in a PCR tube according to the reaction system shown in Table 1 (wherein the amplification primers and probe sequences are as shown in Table 2, that is, the amplification primers can form a completely complementary hairpin structure), and the RT-qPCR reaction was performed on a Bio-rad PCR instrument.
  • the reaction procedure was as follows: 42°C for 30 min; 94°C for 2 min; 94°C for 30 s, 65°C for 30 s, 72°C for 5 min, 40 cycles; 72°C for 5 min; 12°C for ⁇ .
  • primer/probes were mixed at a concentration of 20 ⁇ M for each primer/probe to obtain a primer/probe pool with a total concentration of 20 ⁇ M;
  • the underlined bases are ribonucleic acid bases, i.e., the corresponding nucleotides are ribonucleotides (rNTP);
  • FAM is a fluorescent group, and BHQ1 is a quenching group;
  • the bold parts in the above primers are specific sequences (upstream specific sequences or downstream specific sequences).
  • Comparative Example 1 A method for detecting SARS-COV-2 genes by multiplex RT-qPCR
  • a method for detecting SARS-COV-2 genes by multiplex RT-qPCR comprising the following steps:
  • the RT-qPCR system was configured in a PCR tube according to the reaction system shown in Table 3 (wherein the amplification primers and probe sequences are shown in Table 4), and the RT-qPCR reaction was performed on a Bio-rad PCR instrument.
  • the reaction procedure was as follows: 42°C for 30 min; 94°C for 2 min; 94°C for 30 s, 65°C for 30 s, 72°C for 5 min, 40 cycles; 72°C for 5 min; 12°C for ⁇ .
  • primer/probes were mixed at a concentration of 20 ⁇ M for each primer/probe to obtain a primer/probe pool with a total concentration of 20 ⁇ M; FAM is the fluorescent group and BHQ1 is the quenching group.
  • Example 1 and Comparative Example 1 were used to detect COVID-19 standards of different concentrations, and then fluorescence detection and electrophoresis detection of the amplified products were performed.
  • the fluorescence detection results of the method of Example 1 and Comparative Example 1 are shown in Table 5 and Figure 3: When the concentration of the standard is 100, 1000 copies/ml When the concentration of the standard is 10 copies/ml, the CT value of the method in Example 1 is 39.3, while the method in Comparative Example 1 cannot detect it. It can be seen that compared with the method in Comparative Example 1, the method in Example 1 can greatly improve the efficiency of PCR amplification and the sensitivity of detection.
  • the amplification primers in Table 1 have one or more partially complementary hairpin structures (for example, the 3' ends of N1-F, N2-F, N3-F, Orf1ab-F, and RDRP-F are reduced by 3 nucleotides respectively), they have the same effect as Example 1.

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Abstract

本发明提供一种多重RT-PCR扩增方法，通过将引物设计为完全或部分互补的发卡结构，使引物3'端在RT过程中丧失结合和延伸的能力，并且引物的5'端链包含一个或多个被特异性切割剂切割的切割位点，从而在RT过程中引物的5'端链的切割位点被特异性切割剂切割，但维持互补双链；在PCR的高温过程中，被切割的5'端链游离，释放出3'端链作为靶标扩增的引物，重新获得3'端引物和模板结合和延伸的能力。

Description

一种多重RT-PCR扩增方法及其应用 技术领域
本发明属于多重RT-PCR扩增技术领域，具体涉及一种多重RT-PCR扩增方法及其应用。
背景技术
近年来，分子检测技术飞速发展，广泛应用于包括肿瘤、遗传病、病原微生物检测等领域。随着各种疾病生物标志物的发现，越来越多的生物标准物用于疾病的诊断、治疗和预后。如ALK-EML基因融合可用于非小型肺癌的靶向药物治疗，micro RNA、ctDNA可以用于肿瘤的早期筛查和诊断，hsp65序列可用于结核分支杆菌的鉴定和分型等。
目前最常用的分子检测技术是RT-qPCR，其操作简单、检测封闭等优点被临床使用，但由于RT-qPCR一次检测的靶标较少，限制了其的应用范围。高分辨熔解曲线的发明使RT-qPCR在一管中实现多个靶标的检测成为了可能，大大拓展了RT-qPCR的应用范围。但对于多重RT-qPCR的扩增反应却并不容易，最大的难点在于RT步骤和PCR步骤是在一个反应，在RT过程中的副产物会严重影响后续的PCR扩增，主要表现在：1)特异性引物在RT过程中容易和模板非特异性结合，产生非特异性产物，并且在后续的PCR扩增中进行指数积累，即在多靶标的RT-qPCR的RT反应中，不同特异性引物3端和模板会有局部的匹配，RT酶会进行会在局部匹配的情况下发生延伸反应，导致非特异性的产生，并在后续的PCR反应中指数放大；2)特异性引物在RT过程中容易互相结合并延伸，形成二聚体，在后续PCR过程中指数积累，导致整个反应的提前终止，即在多靶标的RT-qPCR的RT反应中，不同特异性引物3端之间会有局部的匹配，逆转录酶会进行会在局部匹配的情况下发生延伸反应，产生二聚体，在后续的PCR过程中指数放大。因此，开发一个多靶标的RT-qPCR试剂盒往往需要经过大量的引物设计、调整和优化，整个过程繁琐复杂，随着靶标的数量增加，其优化难度往往也指数增加。因此，急需一种可以有效避免RT-PCR中的RT反应中的非特异性扩增和二聚体生成的多重RT-PCR扩增方法。
发明内容
本发明第一方面的目的，在于提供RT-PCR引物。
本发明第二方面的目的，在于提供一种试剂盒。
本发明第三方面的目的，在于提供一种多重RT-PCR扩增方法。
为了实现上述目的，本发明所采取的技术方案是：
本发明的第一个方面，提供RT-PCR引物，包含：上游引物和下游引物；
所述上游引物从5’端到3’端依次包含：第一序列和第二序列：
g1)所述第一序列为与上游特异性序列完全互补的序列，所述第二序列与所述第一序列完全互补(即第二序列为上游特异性序列)，即所述上游引物呈完全互补的发卡结构；或
g2)所述第一序列为与上游特异性序列完全互补的序列，所述第二序列与所述第一序列部分互补(即第二序列为上游特异性序列的部分序列(优选为上游特异性序列的5’端序列))，即所述上游引物呈3’端部分互补的发卡结构(即具有5’突出端的发卡结构)；或
g3)所述第二序列为上游特异性序列，所述第一序列与所述第二序列部分互补(即第一序列为与上游特异性序列的部分序列互补的序列(优选为与上游特异性序列的5’端序列互补的序列))，即所述上游引物呈5’端部分互补的发卡结构(即具有3’突出端的发卡结构)；
所述下游引物从5’端到3’端依次包含：第三序列和第四序列：
h1)所述第三序列为与下游特异性序列完全互补的序列，所述第四序列与所述第三序列完全互补(即 第四序列为下游特异性序列)，即所述下游引物呈完全互补的发卡结构；或
h2)所述第三序列为与下游特异性序列完全互补的序列，所述第四序列与所述第三序列部分互补(即第四序列为下游特异性序列的部分序列(优选为下游特异性序列的5’端序列))，即所述下游引物呈3’端部分互补的发卡结构(即具有5’突出端的发卡结构)；或
h3)所述第四序列为下游特异性序列，所述第三序列与所述第四序列部分互补(即第三序列为与下游特异性序列的部分序列互补的序列(优选为与下游特异性序列的5’端序列互补的序列))，即所述下游引物呈5’端部分互补的发卡结构(即具有3’突出端的发卡结构)；
所述上游引物的5’端链包含一个或多个可以被特异性切割剂切割的切割位点；
所述下游引物的5’端链包含一个或多个可以被特异性切割剂切割的切割位点。
优选地，所述特异性切割剂包含酶促切割剂。
优选地，所述酶促切割剂包含核糖核酸酶(例如：核糖核酸酶H)、糖基化酶(例如：尿嘧啶-DNA糖基化酶(UDG)、USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)、3-甲基腺嘌呤DNA糖基化酶、3-甲基腺嘌呤DNA糖基化酶II、嘧啶水合物-DNA糖基化酶、FaPy-DNA糖基化酶、胸腺嘧啶错配-DNA糖基化酶(例如：次黄嘌呤-DNA糖基化酶、尿嘧啶DNA糖基化酶(UDG)、5-羟甲基尿嘧啶DNA糖基化酶(HmUDG)、5-羟甲基胞嘧啶DNA糖基化酶、1,N6-亚乙烯基腺嘌呤DNA糖基化酶))中的至少一种。
优选地，所述切割位点包含c1)～c2)中至少一种：
c1)核糖核苷酸(rNTP，例如：rATP、rGTP、tCTP、rUTP)；
c2)尿嘧啶脱氧核糖核苷酸(dUTP)。
进一步优选地，所述切割位点包含c1)。
进一步优选地，所述酶促切割剂包含核糖核酸酶(例如：核糖核酸酶H)、糖基化酶(例如：尿嘧啶-DNA糖基化酶(UDG)、USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)中的至少一种；更进一步为核糖核酸酶(例如：核糖核酸酶H)。
优选地，所述核糖核酸酶H包含核糖核酸酶HⅠ、核糖核酸酶HII中至少一种。
优选地，所述上游引物的5’端链包含多个可以被特异性切割剂切割的切割位点，切割位点大于N/10碱基，例如：可以是切割位点的个数大于等于(优选大于)n个；或者是n～N个；其中，上述n＝N/10，N为第一序列的核苷酸个数；或者是3～6个。
优选地，所述下游引物的5’端链包含多个可以被特异性切割剂切割的切割位点，切割位点大于N/10碱基，例如：可以是切割位点的个数大于等于(优选大于)n个；或者是n～N个；其中，上述n＝N/10，N为第三序列的核苷酸个数；或者是3～6个。
优选地，所述可以被特异性切割剂切割的切割位点的个数为整数。
优选地，所述多个可以被特异性切割剂切割的切割位点在所述上游引物的5’端链中随机分布。
优选地，所述多个可以被特异性切割剂切割的切割位点在所述下游引物的5’端链中随机分布。
优选地，所述第一序列与第二序列直接或间接连接。
优选地，所述第一序列与第二序列通过以下任意一种方式连接：碱基连接、linker连接(比如：间臂(spacer3、spacer6、spacer9、spacer18等))。
优选地，所述第三序列与第四序列直接或间接连接。
优选地，所述第三序列与第四序列通过以下任意一种方式连接：碱基连接、linker连接(比如：间臂(spacer3、spacer6、spacer9、spacer18等))。
优选地，所述上游引物的5’端链包含第一序列。
优选地，所述下游引物的5’端链包含第三序列。
优选地，所述第一序列的核苷酸个数为20～60。
优选地，所述第三序列的核苷酸个数为20～60。
优选地，所述第二序列的核苷酸个数为5～65。
优选地，所述第四序列的核苷酸个数为5～65。
优选地，所述上游引物呈3’端部分互补的发卡结构时，所述第一序列与第二序列的核苷酸个数的差值为1～55。
优选地，所述上游引物呈5’端部分互补的发卡结构时，所述第二序列与第一序列的核苷酸个数的差值为3～10。
优选地，所述下游引物呈3’端部分互补的发卡结构时，所述第三序列与第四序列的核苷酸个数的差值为1～55。
优选地，所述下游引物呈5’端部分互补的发卡结构时，所述第四序列与第三序列的核苷酸个数的差值为3～10。
优选地，所述切割位点包含可以被特异性切割剂切割的核苷酸。
优选地，所述上游引物和下游引物中所述的特异性切割剂可以相同或不同。
优选地，所述上游引物中的多个特异性切割剂可以相同或不同。
优选地，所述下游引物中的多个特异性切割剂可以相同或不同。
优选地，所述上游引物和下游引物中所述的切割位点可以相同或不同。
优选地，所述上游引物中的多个切割位点可以相同或不同。
优选地，所述下游引物中的多个切割位点可以相同或不同。
优选地，所述上游特异性序列和下游特异性序列分别与目标区域的两个区段相同或互补。
优选地，所述RT-PCR引物包含多对RT-PCR引物。
本发明的第二个方面，提供一种试剂盒，包含：本发明第一个方面的RT-PCR引物。
优选地，所述试剂盒还包含：本发明第一个方面中所述特异性切割剂。
优选地，所述试剂盒还包含：具有3’-5’核酸外切酶活性的DNA聚合酶(例如：T4DNA聚合酶、DNA聚合酶I(Klenow)大片段、DNA聚合酶I(E.coli))。
优选地，所述试剂盒还包含：逆转录酶。
优选地，所述试剂盒还包含：逆转录引物。
优选地，所述逆转录引物为6～12nt的随机引物；进一步为随机引物(N6)。
优选地，所述逆转录酶包含AMV逆转录酶、M-MuLV逆转录酶中的至少一种。
优选地，所述试剂盒包含：核酸提取试剂组合。
优选地，所述核酸提取试剂组合具体是用于选自以下任意一种方法的核酸提取试剂组合：碱裂解法、酚氯仿抽提法、螯合树脂法、离心柱膜吸附法以及磁珠法。
优选地，所述核酸提取试剂组合包含：裂解液、洗涤液、洗脱液、核酸吸附物中的至少一种；进一步优选地，所述核酸提取试剂组合包含：裂解液、洗涤液、洗脱液和核酸吸附物。
优选地，所述核酸吸附物包含磁珠、吸附膜中的至少一种。
优选地，所述试剂盒用于d1)～d3)中任一种：
d1)扩增目标区域；
d2)检测目标区域；
d3)构建目标区域的文库(构建靶向文库)。
优选地，所述试剂盒用于检测目标区域时，所述试剂盒还包含：检测探针、核酸染料中的至少一种。
优选地，所述检测探针包含TaqMan探针、分子信标、双杂交探针、复合探针中的至少一种；进一步 为TaqMan探针。
优选地，所述核酸染料包含EtBr、SYBR Green、SYBR Gold、GelRed、GelGreen中的至少一种。
本发明的第三个方面，提供一种多重RT-PCR扩增方法，包含采用本发明第一个方面的RT-PCR引物的步骤。
优选地，所述多重RT-PCR扩增方法包括如下步骤：利用本发明第一个方面的RT-PCR引物对待测样本的RNA进行多重RT-PCR反应。
优选地，所述反应进一步包括：
用所述特异性切割剂处理所述多重RT-PCR反应的体系。
优选地，所述多重RT-PCR反应的逆转录(RT)反应与扩增(PCR)反应在同一体系中进行。
优选地，所述同一体系具体为在同一反应体系中进行；即无需先在一个反应体系中进行逆转录反应，然后再在其他反应体系中进行扩增反应。
优选地，所述第一序列和第二序列为g2)、和/或所述第三序列和第四序列为h2)时，所述多重RT-PCR反应还包含所述上游引物和/或所述下游引物延伸的步骤，得到完全互补的发卡结构的上游引物和/或下游引物。
优选地，所述上游引物和/或所述下游引物延伸的步骤与所述多重RT-PCR反应的RT反应同时进行。
优选地，用所述特异性切割剂处理所述多重RT-PCR的反应体系的方法可以是：
使所述多重RT-PCR反应的体系包含所述特异性切割剂。
优选地，用所述特异性切割剂处理所述多重RT-PCR反应的体系的步骤在所述多重RT-PCR反应的RT反应过程中或所述多重RT-PCR反应的RT反应后进行。
优选地，所述多重RT-PCR反应的体系还包含：具有3’-5’核酸外切酶活性的DNA聚合酶(优选本发明第二个方面中的具有3’-5’核酸外切酶活性的DNA聚合酶)。
优选地，所述多重RT-PCR反应的体系还包含：逆转录酶。
优选地，所述多重RT-PCR反应的体系还包含：逆转录引物。
优选地，所述RNA通过所述待测样本发生裂解反应得到。
优选地，所述裂解反应在所述多重RT-PCR反应之前。
优选地，所述裂解反应和所述多重RT-PCR反应在同一体系中进行。
优选地，所述同一体系具体为同一反应容器。
优选地，所述裂解反应后和所述多重RT-PCR反应前不包含：提取纯化步骤。
优选地，所述裂解反应后和所述多重RT-PCR反应前还包含：提取纯化步骤。
优选地，所述提取纯化进一步包含：沉淀核酸或吸附核酸。
优选地，所述提取纯化后还包含：洗脱或溶解核酸。
优选地，所述裂解反应和所述多重RT-PCR反应在不同体系中进行，即不在同一体系中进行。
优选地，所述同一体系具体为同一反应容器。
优选地，所述裂解反应后还包含：提取纯化步骤。
优选地，所述提取纯化进一步包含：沉淀核酸或吸附核酸。
优选地，所述提取纯化后还包含：洗脱或溶解核酸。
优选地，所述裂解的方法包含：物理方式、化学方式、生物方式中的至少一种。
优选地，所述多重RT-PCR反应的体系中还包含检测探针、核酸染料中的至少一种。
优选地，所述检测探针包含TaqMan探针、分子信标、双杂交探针、复合探针中的至少一种；
优选地，所述核酸染料包含EtBr、SYBR Green、SYBR Gold、GelRed、GelGreen中的至少一种。
本发明的有益效果是：
本发明提供了RT-PCR引物，发明人创造性地将引物(上游引物和/或下游引物)设计为完全互补或部分互补的发卡结构(3’端部分互补的发卡结构或5’端部分互补的发卡结构)，从而使引物3’端在RT过程中丧失结合能力(不与模板发生非特异性结合和/或避免引物之间结合)和延伸能力，并且该引物的5’端链包含一个或多个可以被特异性切割剂切割的切割位点，从而在RT-PCR的RT过程中引物的5’端链的切割位点被特异性切割剂切割(对于3’端部分互补的发卡结构还包含引物在逆转录酶和/或DNA聚合酶的作用下延伸得到完全互补的发卡结构的步骤)，但在RT过程中维持互补的双链状态；在后续的PCR的高温过程(例如热变性)中，被切割的5’端链游离，释放出3’端链作为目标区域扩增的引物，重新获得引物3’端与模板结合和延伸的能力；采用本发明的RT-PCR引物可以实现一步法扩增，并且可以有效避免RT过程中多对引物在逆转录酶体系的作用下形成二聚体，以及多对引物在逆转录酶体系的作用下和模板非特异性结合，产生非特异性产物，大幅度提高PCR扩增的效率以及检测的灵敏度和特异性。
附图说明
图1是本发明的引物的结构示意图。
图2是本发明的引物进行RT-PCR扩增的原理图。
图3是实施例1和对比例1的荧光检测结果图。
图4是实施例1和对比例1的扩增产物的电泳图。
具体实施方式
在本发明的描述中，术语“第一”、“第二”、“第三”、“第四”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”、“第三”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
本发明的第一个方面，提供RT-PCR引物，包含：上游引物和下游引物；
所述上游引物从5’端到3’端依次包含：第一序列和第二序列：
g1)所述第一序列为与上游特异性序列完全互补的序列，所述第二序列与所述第一序列完全互补(即第二序列为上游特异性序列)，即所述上游引物呈完全互补的发卡结构；或
g2)所述第一序列为与上游特异性序列完全互补的序列，所述第二序列与所述第一序列部分互补(即第二序列为上游特异性序列的部分序列(优选为上游特异性序列的5’端序列))，即所述上游引物呈3’端部分互补的发卡结构(即具有5’突出端的发卡结构)；或
g3)所述第二序列为上游特异性序列，所述第一序列与所述第二序列部分互补(即第一序列为与上游特异性序列的部分序列互补的序列(优选为与上游特异性序列的5’端序列互补的序列))，即所述上游引物呈5’端部分互补的发卡结构(即具有3’突出端的发卡结构)；
所述下游引物从5’端到3’端依次包含：第三序列和第四序列：
h1)所述第三序列为与下游特异性序列完全互补的序列，所述第四序列与所述第三序列完全互补(即第四序列为下游特异性序列)，即所述下游引物呈完全互补的发卡结构；或
h2)所述第三序列为与下游特异性序列完全互补的序列，所述第四序列与所述第三序列部分互补(即第四序列为下游特异性序列的部分序列(优选为下游特异性序列的5’端序列))，即所述下游引物呈3’端部分互补的发卡结构(即具有5’突出端的发卡结构)；或
h3)所述第四序列为下游特异性序列，所述第三序列与所述第四序列部分互补(即第三序列为与下游特异性序列的部分序列互补的序列(优选为与下游特异性序列的5’端序列互补的序列))，即所述下游引物呈5’端部分互补的发卡结构(即具有3’突出端的发卡结构)；
所述上游引物的5’端链包含一个或多个可以被特异性切割剂切割的切割位点；
所述下游引物的5’端链包含一个或多个可以被特异性切割剂切割的切割位点。
对于上述RT-PCR引物中的第一序列、第二序列、第三序列和第四序列，存在以下九种组合：
a1)：g1)和h1)；a2)：g1)和h2)；a3)：g1)和h3)；a4)：g2)和h1)；a5)：g2)和h2)；a6)：g2)和h3)；a7)：g3)和h1)；a8)：g3)和h2)；a9)：g3)和h3)。
优选地，所述第一序列为与上游特异性序列完全互补的序列，所述第二序列与所述第一序列完全互补(即第二序列为上游特异性序列)，所述第三序列为与下游特异性序列完全互补的序列，所述第四序列与所述第三序列完全互补(即第四序列为下游特异性序列)。
在多重RT-PCR过程中，RT过程中的副产物严重影响了PCR过程，主要表现在：b1)不同特异性引物3’端和模板会有局部的匹配，逆转录酶会进行会在局部匹配的情况下发生延伸反应，导致非特异性产物的产生，并在后续的PCR反应中指数放大；b2)不同特异性引物3’端之间会有局部的匹配，逆转录酶会进行会在局部匹配的情况下发生延伸反应，产生二聚体，在后续的PCR过程中指数放大；
在本发明中，发明人创造性地将引物(上游引物和/或下游引物)设计为完全互补或部分互补的发卡结构(3’端部分互补的发卡结构或5’端部分互补的发卡结构)，从而使引物3’端在RT过程中丧失结合能力(不与模板发生非特异性结合和/或避免引物之间结合)和延伸能力，并且该引物的5’端链包含一个或多个可以被特异性切割剂切割的切割位点(图1)，从而在RT-PCR的RT过程中引物的5’端链的切割位点被特异性切割剂切割(对于3’端部分互补的发卡结构的引物还包含引物在逆转录酶和/或DNA聚合酶的作用下延伸得到完全互补的发卡结构的引物的步骤)，但在RT过程中维持互补的双链状态；在后续的PCR的高温过程(例如热变性)中，被切割的5’端链游离，释放出3’端链作为目标区域扩增的引物，重新获得引物3’端与模板结合和延伸的能力，对于完全互补或部分互补的发卡结构的引物的扩增原理(图2)具体如下：
e1)引物结构为完全互补的发卡结构：特异性序列(上游特异性序列和/或下游特异性序列，对应上述的第二序列和第四序列)和与其完全互补的寡核苷酸链(例如DNA链，对应上述的第一序列和第三序列)形成“封闭”发卡结构，可以阻止引物在RT过程中和模板非特异性结合以及引物之间的结合，其中，引物的5’端链包含一个或多个被可以被特异性切割剂切割的切割位点，从而在RT-PCR的RT过程中引物的5’端链的切割位点被特异性切割剂切割，但在RT过程中维持互补的双链状态；在后续的PCR的高温过程(例如热变性)中，被切割的5’端链游离，释放出3’端链作为目标区域扩增的引物；
e2)引物结构为部分互补的发卡结构：
e21)当引物结构为3’端部分互补的发卡结构时，与特异性序列(上游特异性序列和/或下游特异性序列)互补的寡核苷酸链(对应上述的第一序列和第三序列)和部分特异性序列(特异性序列的5’端序列，对应上述的第二序列和第四序列)形成3’端部分互补的发卡结构(即具有5’突出端的发卡结构)，其中，引物的5’端链包含一个或多个可以被特异性切割剂切割的切割位点；首先，RT过程中，引物在逆转录酶和/或DNA聚合酶的作用下延伸，得到包含特异性序列全序列的引物，并形成完全互补的发卡结构；并且，引物的5’端链的切割位点被特异性切割剂切割，但在RT过程中维持互补的双链状态；在后续的PCR的高温过程(例如热变性)中，被切割的5’端链游离，释放出3’端链作为目标区域扩增的引物；
e22)当引物结构为5’端部分互补的发卡结构时，特异性序列(上游特异性序列和/或下游特异性序列，对应上述的第二序列和第四序列)和与其部分序列互补的寡核苷酸链(优选与特异性序列的5’端序列互补的序列，例如DNA链，对应上述的第一序列和第三序列)形成5’端部分互补的发卡结构，可以阻止引物在RT过程中和模板非特异性结合以及引物之间的结合，其中，引物的3’突出3个碱基以上的末端可以防止被逆转录酶非特异性加上碱基或者可以被具有3’-5’核酸外切酶活性的DNA聚合酶切割成平末端，且引物的5’端链包含一个或多个被可以被特异性切割剂切割的切割位点，从而在RT-PCR的RT过程中引物的5’端链的切割位点被特异性切割剂切割，但在RT过程中维持部分互补或完全互补的双链状态；在后续的PCR的高温过程(例如热变性)中，被切割的5’端链游离，释放出3’端链作为目标区域扩增的引物。
采用本发明的RT-PCR引物可以实现一步法扩增，并且可以有效避免RT过程中多对引物在逆转录酶 体系的作用下形成二聚体，以及多对引物在逆转录酶体系的作用下和模板非特异性结合，产生非特异性产物，大幅度提高PCR扩增的效率以及检测的灵敏度和特异性。
优选地，所述上游特异性序列和下游特异性序列分别与目标区域的两个区段相同或互补。
优选地，所述引物(上游引物和/或下游引物)中的特异性序列(上游特异性序列和/或下游特异性序列)的设计遵循常规的引物设计原则，比如：上游特异性序列与目标区域上游负链(即反义链、cDNA第二链)序列互补或正链(即有义链、cDNA第一链)序列相同，下游特异性序列与目标区域的下游正链(即有义链、cDNA第一链)序列互补或负链(即反义链、cDNA第二链)序列相同。
优选地，所述上游特异性序列与目标区域的上游负链序列互补或正链序列相同。
优选地，所述下游特异性序列与目标区域的下游正链序列互补或负链序列相同。
优选地，所述特异性切割剂包含酶促切割剂。
优选地，所述酶促切割剂包含核糖核酸酶(例如：核糖核酸酶H)、糖基化酶(例如：尿嘧啶-DNA糖基化酶(UDG)、USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)、3-甲基腺嘌呤DNA糖基化酶、3-甲基腺嘌呤DNA糖基化酶II、嘧啶水合物-DNA糖基化酶、FaPy-DNA糖基化酶、胸腺嘧啶错配-DNA糖基化酶(例如：次黄嘌呤-DNA糖基化酶、尿嘧啶DNA糖基化酶(UDG)、5-羟甲基尿嘧啶DNA糖基化酶(HmUDG)、5-羟甲基胞嘧啶DNA糖基化酶、1,N6-亚乙烯基腺嘌呤DNA糖基化酶))中的至少一种。
优选地，所述切割位点包含c1)～c2)中至少一种：
c1)核糖核苷酸(rNTP，例如：rATP、rGTP、tCTP、rUTP)，其对应的特异性切割剂为核糖核酸酶(例如：核糖核酸酶H)，当rNTP为rUTP时，其对应的特异性切割剂也可以是尿嘧啶-DNA糖基化酶(UDG)或USER酶(尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)；
c2)尿嘧啶脱氧核糖核苷酸(dUTP)，其对应的特异性切割剂是尿嘧啶-DNA糖基化酶(UDG)或USER酶(尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)。
进一步优选地，所述切割位点包含c1)。
进一步优选地，所述酶促切割剂包含核糖核酸酶(例如：核糖核酸酶H)、糖基化酶(例如：尿嘧啶-DNA糖基化酶(UDG)、USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)中的至少一种；更进一步为核糖核酸酶(例如：核糖核酸酶H)。
优选地，所述核糖核酸酶H包含核糖核酸酶HⅠ、核糖核酸酶HII中至少一种。
优选地，所述上游引物和下游引物中所述的特异性切割剂可以相同或不同。
优选地，所述上游引物中的多个特异性切割剂可以相同或不同。
优选地，所述下游引物中的多个特异性切割剂可以相同或不同。
优选地，所述上游引物和下游引物中所述的切割位点可以相同或不同。
优选地，所述上游引物中的多个切割位点可以相同或不同。
优选地，所述下游引物中的多个切割位点可以相同或不同。
优选地，所述上游引物的5’端链包含多个可以被特异性切割剂切割的切割位点，切割位点大于N/10碱基，例如：可以是切割位点的个数大于等于(优选大于)n个；或者是n～N个；其中，上述n＝N/10，N为第一序列的核苷酸个数；或者是3～6个。
优选地，所述下游引物的5’端链包含多个可以被特异性切割剂切割的切割位点，，切割位点大于N/10碱基，例如：可以是切割位点的个数大于等于(优选大于)n个；或者是n～N个，其中，上述n＝N/10，N为第三序列的核苷酸个数；或者是3～6个。
优选地，所述可以被特异性切割剂切割的切割位点的个数为整数。
优选地，所述多个可以被特异性切割剂切割的切割位点在所述上游引物的5’端链中随机分布。
优选地，所述多个可以被特异性切割剂切割的切割位点在所述下游引物的5’端链中随机分布。
优选地，所述第一序列与第二序列直接或间接连接。
优选地，所述第一序列与第二序列通过以下任意一种方式连接：碱基连接、linker连接(比如：间臂(spacer3、spacer6、spacer9、spacer18等))。
优选地，所述第三序列与第四序列直接或间接连接。
优选地，所述第三序列与第四序列通过以下任意一种方式连接：碱基连接、linker连接(比如：间臂(spacer3、spacer6、spacer9、spacer18等))。
优选地，所述上游引物的5’端链包含第一序列。
优选地，所述下游引物的5’端链包含第三序列。
优选地，所述第一序列的核苷酸个数为20～60。
优选地，所述第三序列的核苷酸个数为20～60。
优选地，所述第二序列的核苷酸个数为5～65。
优选地，所述第四序列的核苷酸个数为5～65。
优选地，所述上游引物呈3’端部分互补的发卡结构时，所述第一序列与第二序列的核苷酸个数的差值为1～55。
优选地，所述上游引物呈5’端部分互补的发卡结构时，所述第二序列与第一序列的核苷酸个数的差值为3～10。
优选地，所述下游引物呈3’端部分互补的发卡结构时，所述第三序列与第四序列的核苷酸个数的差值为1～55。
优选地，所述下游引物呈5’端部分互补的发卡结构时，所述第四序列与第三序列的核苷酸个数的差值为3～10。
优选地，所述切割位点包含可以被特异性切割剂切割的核苷酸。
优选地，所述RT-PCR引物为DNA片段或其类似物。
优选地，所述RT-PCR引物包含多对RT-PCR引物。
优选地，所述多对为两对以上。
优选地，检测SARS-COV-2基因的RT-PCR引物，包含：上游引物和下游引物；
所述上游引物的序列如SEQ ID NO.1、4、7、10、13所示，所述下游引物的序列如SEQ ID NO.2、5、8、11、14所示；
序列如SEQ ID NO.1、4、7、10、13所示的上游引物的5’端各自独立包含一个或多个可以被特异性切割剂切割的切割位点；
序列如SEQ ID NO.2、5、8、11、14所示的下游引物的5’端各自独立包含一个或多个可以被特异性切割剂切割的切割位点。
优选地，所述特异性切割剂包含酶促切割剂。
优选地，所述酶促切割剂包含核糖核酸酶(例如：核糖核酸酶H)、糖基化酶(例如：尿嘧啶-DNA糖基化酶(UDG)、USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)、3-甲基腺嘌呤DNA糖基化酶、3-甲基腺嘌呤DNA糖基化酶II、嘧啶水合物-DNA糖基化酶、FaPy-DNA糖基化酶、胸腺嘧啶错配-DNA糖基化酶(例如：次黄嘌呤-DNA糖基化酶、尿嘧啶DNA糖基化酶(UDG)、5-羟甲基尿嘧啶DNA糖基化酶(HmUDG)、5-羟甲基胞嘧啶DNA糖基化酶、1,N6-亚乙烯基腺嘌呤DNA糖基化酶))中的至少一种。
优选地，所述切割位点包含c1)～c2)中至少一种：
c1)核糖核苷酸(rNTP，例如：rATP、rGTP、tCTP、rUTP)，其对应的特异性切割剂为核糖核酸酶(例 如：核糖核酸酶H)，当rNTP为rUTP时，其对应的特异性切割剂也可以是尿嘧啶-DNA糖基化酶(UDG)或USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)；
c2)尿嘧啶脱氧核糖核苷酸(dUTP)，其对应的特异性切割剂是尿嘧啶-DNA糖基化酶(UDG)或USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)。
进一步优选地，所述切割位点包含c1)。
进一步优选地，所述酶促切割剂包含核糖核酸酶(例如：核糖核酸酶H)、糖基化酶(例如，尿嘧啶-DNA糖基化酶(UDG)、USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)中的至少一种；更进一步为核糖核酸酶(例如：核糖核酸酶H)。
优选地，所述核糖核酸酶H包含核糖核酸酶HⅠ、核糖核酸酶HII中至少一种。
优选地，序列如SEQ ID NO.1、4、7、10、13所示的上游引物的核糖核苷酸的位置如表1及附注所示。
优选地，序列如SEQ ID NO.2、5、8、11、14所示的下游引物的核糖核苷酸的位置如表2及附注所示。
优选地，所述RT-PCR引物用于待测样本的RNA的扩增。
优选地，所述待测样本包含细胞、组织、体液、微生物、唾液、尿液、痰液、粪便、咽拭子、鼻拭子。
优选地，所述体液包含组织液、淋巴液、血液、脑脊液中的至少一种。
优选地，所述微生物包含细菌、病毒、真菌、放线菌、立克次氏体、支原体、衣原体、螺旋体中的至少一种。
优选地，所述RNA包含mRNA、lncRNA、small RNA、cfRNA和tRNA中的至少一种。
优选地，所述small RNA可为miRNA、siRNA和piRNA中的至少一种。
本发明的第二个方面，提供一种试剂盒，包含：本发明第一个方面的RT-PCR引物。
优选地，所述试剂盒还包含：所述特异性切割剂，其用于RT-PCR的RT过程中完全互补或部分互补的发卡结构的引物(上游引物和/或下游引物)的5’端链的切割位点的切割。
优选地，所述试剂盒还包含：具有3’-5’核酸外切酶活性的DNA聚合酶(例如：T4DNA聚合酶、DNA聚合酶I(Klenow)大片段、DNA聚合酶I(E.coli))，其用于保持所述引物(上游引物和/或下游引物)3’端平末端结构(抑制逆转录酶的末端转移活性)，该DNA聚合酶也可以用于引物(上游引物和/或下游引物)延伸，得到包含特异性序列全序列的引物，并形成完全互补的发卡结构(所述第二序列与所述第一序列部分互补和/或所述第四序列与所述第三序列部分互补时)。
优选地，所述试剂盒还包含：逆转录反应混合液，其用于逆转录反应(即将RNA逆转录为cDNA)。
优选地，所述逆转录反应混合液包含：逆转录酶、DNA聚合酶、逆转录缓冲液、逆转录引物、dNTPs、RNA酶抑制剂中的至少一种；优选地，所述逆转录反应混合液包含：逆转录酶、DNA聚合酶、逆转录缓冲液、dNTPs、RNA酶抑制剂和逆转录引物；其中的逆转录酶和/或DNA聚合酶也可以用于引物(上游引物和/或下游引物)延伸，得到包含特异性序列全序列的引物，并形成完全互补的发卡结构(所述引物结构为3’端部分互补的发卡结构时)；对于试剂盒中已经包含的组分，所述逆转录反应混合液可以相应地减少该组分：比如：所述试剂盒包含具有3’-5’核酸外切酶活性的DNA聚合酶时，所述逆转录反应混合液可以不包含DNA聚合酶。
优选地，所述逆转录引物为6～12nt的随机引物；进一步为随机引物(N6)。
优选地，所述逆转录酶包含AMV逆转录酶、M-MuLV逆转录酶中的至少一种。
优选地，所述试剂盒还包含：PCR反应混合液，其用于PCR反应。
优选地，所述PCR反应混合液包含：DNA聚合酶、Mg2+、PCR缓冲液、dNTPs中的至少一种；进一步优选地，所述PCR反应混合液还包含：DNA聚合酶、Mg2+、PCR缓冲液和dNTPs；其中的DNA聚合 酶也可以用于引物(上游引物和/或下游引物)延伸，得到包含特异性序列全序列的引物，并形成完全互补的发卡结构(所述引物结构为3’端部分互补的发卡结构时)；对于试剂盒已经包含的组分，所述PCR反应混合液可以相应地减少该组分，比如：所述试剂盒包含具有3’-5’核酸外切酶活性的DNA聚合酶，和/或所述试剂盒的逆转录反应液中包含DNA聚合酶时，所述PCR反应混合液可以不包含DNA聚合酶。
优选地，所述DNA聚合酶为耐高温DNA聚合酶(例如Taq DNA聚合酶)；进一步为Taq热启动DNA聚合酶。
优选地，所述试剂盒包含：核酸提取试剂组合。
优选地，所述核酸提取试剂组合具体是用于选自以下任意一种方法的核酸提取试剂组合：碱裂解法、酚氯仿抽提法、螯合树脂法、离心柱膜吸附法以及磁珠法。
优选地，所述核酸提取试剂组合包含：裂解液、洗涤液、洗脱液、核酸吸附物中的至少一种；进一步优选地，所述核酸提取试剂组合包含：裂解液、洗涤液、洗脱液和核酸吸附物。
优选地，所述核酸吸附物包含磁珠、吸附膜中的至少一种。
优选地，所述试剂盒用于d1)～d3)中任一种：
d1)扩增目标区域；
d2)检测目标区域；
d3)构建目标区域的文库(构建靶向文库)。
优选地，所述试剂盒用于检测目标区域时，所述试剂盒还包含：检测探针、核酸染料中的至少一种；进一步包含检测探针，所述检测探针是现有RT-qPCR中常用的探针，本领域技术人员可以基于所需要检测的目标区域设计。
优选地，所述检测探针包含TaqMan探针、分子信标、双杂交探针、复合探针中的至少一种；进一步为TaqMan探针。
优选地，所述核酸染料包含EtBr、SYBR Green、SYBR Gold、GelRed、GelGreen中的至少一种。
优选地，所述试剂盒用于检测SARS-COV-2基因时，所述RT-PCR引物为本发明第一个方面的检测SARS-COV-2基因的RT-PCR引物。
优选地，所述试剂盒用于检测SARS-COV-2基因时，所述试剂盒还包含检测SARS-COV-2基因的探针，所述检测SARS-COV-2基因的探针的序列如SEQ ID NO.3、6、9、10、15所示。
优选地，所述检测SARS-COV-2基因的探针的两端分别连接有淬灭基团和荧光基团。
优选地，所述荧光基团包含FAM、TET、VIC、HEX、Cy5、ROX、Texas Red、Tamara、JOE中的至少一种；进一步优选地，所述荧光基团包含FAM。
优选地，所述淬灭基团包含BHQ1、BHQ2、BHQ3、MGB中至少一种；进一步优选地，所述淬灭基团包含BHQ1。
优选地，所述试剂盒用于构建目标区域的文库时，所述试剂盒还包含：用于添加测序接头的试剂。
优选地，所述用于添加测序接头的试剂是选自以下任一种添加测序接头的方法：通过PCR添加测序接头的方法，通过连接酶添加测序接头的方法。
本发明的第三个方面，提供一种测序试剂套装，包含：采用本发明第二个方面的试剂盒。
优选地，所述测序试剂套装还包含：测序试剂盒。
本发明的第四个方面，提供一种测序系统，包含：测序仪和本发明第三个方面的测序试剂套装。
本发明的第五个方面，提供一种多重RT-PCR扩增方法，包含采用本发明第一个方面的RT-PCR引物的步骤。
优选地，所述多重RT-PCR扩增方法包括如下步骤：利用本发明第一个方面的RT-PCR引物对待测样本的RNA进行多重RT-PCR反应。
优选地，所述反应进一步包括：
用所述特异性切割剂处理所述多重RT-PCR反应的体系。
优选地，所述多重RT-PCR反应的逆转录(RT)反应与扩增(PCR)反应在同一体系中进行。
优选地，所述同一体系具体为在同一反应体系中进行；即无需先在一个反应体系中进行逆转录反应，然后再在其他反应体系中进行扩增反应。
优选地，所述第一序列和第二序列为g2)、和/或所述第三序列和第四序列为h2)时，所述多重RT-PCR反应还包含所述上游引物和/或所述下游引物延伸的步骤，得到完全互补的发卡结构的上游引物和/或下游引物。
优选地，所述上游引物和/或所述下游引物延伸的步骤与所述多重RT-PCR反应的RT反应同时进行。
本发明的方法中，通过将引物(上游引物和/或下游引物)设计为完全互补或部分互补的发卡结构(3’端部分互补的发卡结构或5’端部分互补的发卡结构)，从而使引物3’端在RT过程中丧失结合能力(不与模板发生非特异性结合和/或避免引物之间结合)和延伸的能力，并且该引物的5’端链包含一个或多个被可以被特异性切割剂切割的切割位点，从而在RT-PCR的RT过程中引物的5’端链的切割位点被特异性切割剂切割(对于3’端部分互补的发卡结构还包含引物在逆转录酶和/或DNA聚合酶的作用下延伸得到完全互补的发卡结构的步骤)，但在RT过程中维持互补的双链状态；在后续的PCR的高温过程(例如热变性)中，被切割的5’端链游离，释放出3’端链作为目标区域扩增的引物，重新获得3’端引物和模板结合和延伸的能力；从而使得采用本发明的方法可以实现一步法扩增，并且可以有效避免RT过程中多对引物在逆转录酶体系的作用下形成二聚体，以及多对引物在逆转录酶体系的作用下和模板非特异性结合，产生非特异性产物，大幅度提高PCR扩增的效率以及检测的灵敏度和特异性。
优选地，用所述特异性切割剂处理所述多重RT-PCR反应的体系的步骤在所述多重RT-PCR反应的RT反应过程中或所述多重RT-PCR反应的RT反应后进行。
优选地，用所述特异性切割剂处理所述多重RT-PCR反应的体系的方法可以是：
使所述多重RT-PCR反应的体系包含所述特异性切割剂。
优选地，所述多重RT-PCR反应的体系还包含：具有3’-5’核酸外切酶活性的DNA聚合酶(优选本发明第二个方面中的具有3’-5’核酸外切酶活性的DNA聚合酶)，其用于保持所述引物(上游引物和/或下游引物)3’端平末端结构(抑制逆转录酶的末端转移活性)，该DNA聚合酶也可以用于引物(上游引物和/或下游引物)延伸，得到包含特异性序列全序列的引物，并形成完全互补的发卡结构(所述引物结构为3’端部分互补的发卡结构时)。
优选地，所述多重RT-PCR反应的体系还包含：逆转录反应混合液，其用于逆转录反应(即将RNA逆转录为cDNA)。
优选地，所述逆转录反应混合液包含：逆转录酶、DNA聚合酶、逆转录缓冲液、逆转录引物、dNTPs、RNA酶抑制剂中的至少一种；优选地，所述逆转录反应混合液包含：逆转录酶、DNA聚合酶、逆转录缓冲液、dNTPs、RNA酶抑制剂和逆转录引物；其中的逆转录酶和/或DNA聚合酶也可以用于引物(上游引物和/或下游引物)延伸，得到包含特异性序列全序列的引物，并形成完全互补的发卡结构(所述引物结构为3’端部分互补的发卡结构时)；对于RT-PCR反应的体系中已经包含的组分，所述逆转录反应混合液可以相应地减少该组分：比如：所述RT-PCR反应的体系包含具有3’-5’核酸外切酶活性的DNA聚合酶时，所述逆转录反应混合液可以不包含DNA聚合酶。
优选地，所述逆转录引物为6～12nt的随机引物；进一步为随机引物(N6)。
优选地，所述逆转录酶包含AMV逆转录酶、M-MuLV逆转录酶中的至少一种。
优选地，所述多重RT-PCR反应的体系还包含：PCR反应混合液，其用于PCR反应。
优选地，所述PCR反应混合液包含：DNA聚合酶、Mg2+、PCR缓冲液、dNTPs中的至少一种；进一 步优选地，所述PCR反应混合液还包含：DNA聚合酶、Mg2+、PCR缓冲液和dNTPs；其中的DNA聚合酶也可以用于引物(上游引物和/或下游引物)延伸，得到包含特异性序列全序列的引物，并形成完全互补的发卡结构(所述引物结构为3’端部分互补的发卡结构时)；对于RT-PCR反应的体系已经包含的组分，所述PCR反应混合液可以相应地减少该组分，比如：所述RT-PCR反应的体系包含具有3’-5’核酸外切酶活性的DNA聚合酶，和/或所述RT-PCR反应的体系的逆转录反应液中包含DNA聚合酶时，所述PCR反应混合液可以不包含DNA聚合酶。
优选地，所述DNA聚合酶为耐高温DNA聚合酶(例如Taq DNA聚合酶)；进一步为Taq热启动DNA聚合酶。
优选地，所述多重RT-PCR反应的体系中所述特异性切割剂的浓度小于用于延伸的酶(比如：所述DNA聚合酶和/或所述逆转录酶)的浓度，从而使延伸反应先于切割反应。
优选地，所述RNA通过所述待测样本发生裂解反应得到。
优选地，所述裂解反应在所述多重RT-PCR反应之前。
优选地，所述裂解反应和所述多重RT-PCR反应在同一体系中进行。
优选地，所述同一体系具体为同一反应容器。
优选地，所述裂解反应后和所述多重RT-PCR反应前不包含：提取纯化步骤。
优选地，所述裂解反应后和所述多重RT-PCR反应前还包含：提取纯化步骤，去除盐类，有机剂等杂质。
优选地，所述提取纯化进一步包含：沉淀核酸或吸附核酸。
优选地，所述提取纯化后还包含：洗脱或溶解核酸。
优选地，所述裂解反应和所述多重RT-PCR反应在不同体系中进行，即不在同一体系中进行。
优选地，所述同一体系具体为同一反应容器。
优选地，所述裂解反应后还包含：提取纯化步骤，去除盐类，有机剂等杂质。
优选地，所述提取纯化进一步包含：沉淀核酸或吸附核酸。
优选地，所述提取纯化后还包含：洗脱或溶解核酸。
优选地，所述裂解的方法包含：物理方式、化学方式、生物方式中的至少一种。
优选地，所述物理方式包含：煮沸法、玻璃珠法、超声波法、研磨法、冻融法、匀浆法中的至少一种。
优选地，所述化学方式包含：表面活性剂法(SDS法)、碱裂解法中的至少一种。
优选地，所述生物方式包含：酶法，比如通过溶菌酶、蛋白酶K等酶裂解。
本发明的第六个方面，提供一种检测目标区域的方法，包括本发明第五个方面的多重RT-PCR扩增方法的步骤。
优选地，所述多重RT-PCR反应的体系中还包含检测探针和/或核酸染料(优选为本发明第二个方面中的检测探针和/或核酸染料)。
优选地，一种检测SARS-COV-2基因的方法，包括如下步骤：利用本发明第一个方面中的检测SARS-COV-2基因的RT-PCR引物对待测样本的RNA进行多重RT-PCR反应。
优选地，所述RT-PCR反应的体系中还包含本发明第二个方面中的检测SARS-COV-2基因的探针。
优选地，所述方法为非诊断目的方法。
本发明的第七个方面，提供一种构建靶向文库的方法，包含本发明第五个方面的多重RT-PCR扩增的方法的步骤；
所述方法还包含如下步骤：对RT-PCR产物添加测序接头，得到测序文库。
优选地，得到测序文库前添加测序接头后还可以包括环化反应，即对扩增得到的线性文库进行环化。
优选地，所述方法还包括如下步骤：对测序文库进行纯化。
优选地，所述纯化采用磁珠进行。
本发明的第八个方面，提供一种测序方法，包含：本发明第七个方面的构建靶向文库的方法的步骤。
优选地，所述测序方法包括如下步骤：制备文库；测序；
所述制备文库的方法为本发明第七个方面的构建靶向文库的方法。
优选地，所述测序前还包括如下步骤：文库质检。
本发明第九个方面，提供一种获得目标区域基因信息的方法，包含：本发明第八个方面的测序方法的步骤。
优选地，所述获得目标区域基因信息的方法包括如下步骤：测序，获得测序数据；获得目标区域基因信息；所述信息包含序列信息、变异信息中的至少一种；
所述测序的方法为本发明第八个方面的测序方法。
优选地，所述信息包含变异信息时，所述获得目标区域基因信息的方法还包含如下步骤：将获得的测序数据与参考基因组进行比对，确定目标区域基因的变异信息。
优选地，所述变异包括基因融合、SNV、基因缺失、基因插入突变中的至少一种。
优选地，所述方法为非诊断目的方法。
本发明第十个方面，提供本发明第一个方面的RT-PCR引物、第二个方面的试剂盒、第三个方面的测序试剂套装和/或第四个方面的测序系统在f1)～f10)任一项中的应用；
f1)构建靶向文库；
f2)制备用于构建靶向文库的产品；
f3)测序；
f4)制备用于测序的产品；
f5)获得目标区域基因信息；
f6)制备用于获得目标区域基因信息的产品；
f7)多重RT-PCR扩增；
f8)制备用于多重RT-PCR扩增的产品；
f9)检测目标区域；
f10)制备用于检测目标区域的产品。
优选地，f5)、f9)所述应用为非诊断目的应用。
以下通过具体的实施例对本发明的内容作进一步详细的说明。
应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。本实施例中所使用的材料、试剂等，如无特别说明，为从商业途径得到的试剂和材料。
下述实施例/对比例的方法用于检测新冠病毒多个基因序列，包含5对引物，分别扩增5个靶标序列，来增加新冠特异性序列被检出的概率；RT-qPCR试剂采用诺唯赞公司(HiScript II One Step RT-PCR Kit，货号P611-01)，Rnase HII酶来自NEB公司，货号M0288S，klenow酶来自美国NEB公司，货号M0210S。
实施例1一种多重RT-qPCR检测SARS-COV-2基因的方法
一种多重RT-qPCR检测SARS-COV-2基因的方法，包括如下步骤：
在PCR管中按照表1所示反应体系配置RT-qPCR体系(其中，扩增引物和探针序列如表2所示，即扩增引物可以形成完全互补的发卡结构)，在Bio-radPCR仪上进行RT-qPCR反应，反应程序如下：42℃ 30min；94℃ 2min；94℃ 30s，65℃ 30s，72℃ 5min，40cycles；72℃ 5min；12℃ ∞。
表1 RT-qPCR体系

表2扩增引物和探针序列
注：上述引物/探针按照每条引物/探针20μM浓度进行混合，得到总浓度为20μM的引物/探针池；上述下划线碱基为核糖核酸碱基，即对应的核苷酸为核糖核苷酸(rNTP)；FAM为荧光基团，BHQ1为淬灭基团；上述引物中加粗部分为特异性序列(上游特异性序列或下游特异性序列)。
对比例1一种多重RT-qPCR检测SARS-COV-2基因的方法
一种多重RT-qPCR检测SARS-COV-2基因的方法，包括如下步骤：
在PCR管中按照表3所示反应体系配置RT-qPCR体系(其中，扩增引物和探针序列如表4所示)，在Bio-radPCR仪上进行RT-qPCR反应，反应程序如下：42℃ 30min；94℃ 2min；94℃ 30s，65℃ 30s，72℃ 5min，40cycles；72℃ 5min；12℃ ∞。
表3 RT-qPCR体系
表4扩增引物和探针序列
注：上述引物/探针按照每条引物/探针20μM浓度进行混合，得到总浓度为20μM的引物/探针池；FAM为荧光基团，BHQ1为淬灭基团。
效果实施例
分别采用实施例1和对比例1的方法对不同浓度的新冠标准品进行检测，然后进行荧光检测和扩增产物的电泳检测。
1.荧光检测结果
采用实施例1和对比例1的方法的荧光检测结果如表5及图3所示：当标准品浓度为100、1000copies/ml 时，实施例1的方法的CT值(Cycle Threshold Value)小于对比例1，当标准品浓度为10copies/ml时，实施例1的方法的CT值为39.3，而对比例1的方法无法检出；可见，相对于对比例1的方法，实施例1的方法能够大幅度提高PCR扩增的效率以及检测的灵敏度。
表5荧光检测结果
2.电泳检测结果
将实施例1和对比例1的RT-qPCR得到的产物进行电泳，结果如图4所示：与对比例1的方法得到的产物相比，实施例1的方法得到的产物更单一(尤其是标准品浓度低，即低浓度病毒载量时)，可见，实施例1的方法特异性更强。
当表1中的扩增引物存在一个或多个部分互补的发卡结构时(比如：N1-F、N2-F、N3-F、Orf1ab-F、RDRP-F的3’端分别减少3个核苷酸)，其具有与实施例1相当的效果。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (13)

  1. RT-PCR引物，包含：上游引物和下游引物；
    所述上游引物从5’端到3’端依次包含：第一序列和第二序列：
    g1)所述第一序列为与上游特异性序列完全互补的序列，所述第二序列与所述第一序列完全互补；或
    g2)所述第一序列为与上游特异性序列完全互补的序列，所述第二序列与所述第一序列部分互补；或
    g3)所述第二序列为上游特异性序列，所述第一序列与所述第二序列部分互补；
    所述下游引物从5’端到3’端依次包含：第三序列和第四序列：
    h1)所述第三序列为与下游特异性序列完全互补的序列，所述第四序列与所述第三序列完全互补；或
    h2)所述第三序列为与下游特异性序列完全互补的序列，所述第四序列与所述第三序列部分互补；或
    h3)所述第四序列为下游特异性序列，所述第三序列与所述第四序列部分互补；
    所述上游引物的5’端链包含一个或多个可以被特异性切割剂切割的切割位点；
    所述下游引物的5’端链包含一个或多个可以被特异性切割剂切割的切割位点。
  2. 根据权利要求1所述的RT-PCR引物，其特征在于：
    所述特异性切割剂包含酶促切割剂；
    优选地，所述酶促切割剂包含核糖核酸酶(例如：核糖核酸酶H)、糖基化酶(例如：尿嘧啶-DNA糖基化酶(UDG)、USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物)、3-甲基腺嘌呤DNA糖基化酶、3-甲基腺嘌呤DNA糖基化酶II、嘧啶水合物-DNA糖基化酶、FaPy-DNA糖基化酶、胸腺嘧啶错配-DNA糖基化酶(例如：次黄嘌呤-DNA糖基化酶、尿嘧啶DNA糖基化酶(UDG)、5-羟甲基尿嘧啶DNA糖基化酶(HmUDG)、5-羟甲基胞嘧啶DNA糖基化酶、1,N6-亚乙烯基腺嘌呤DNA糖基化酶))中的至少一种。
  3. 根据权利要求2所述的RT-PCR引物，其特征在于：
    所述切割位点包含c1)～c2)中至少一种：
    c1)核糖核苷酸；
    c2)尿嘧啶脱氧核糖核苷酸。
  4. 根据权利要求3所述的RT-PCR引物，其特征在于：
    所述酶促切割剂包含核糖核酸酶(例如：核糖核酸酶H)、糖基化酶(例如：尿嘧啶-DNA糖基化酶(UDG)、USER酶(例如：尿嘧啶DNA糖基化酶(UDG)和DNA糖基化酶-裂解酶Endo VIII的混合物))中的至少一种；进一步包含核糖核酸酶；
    优选地，所述切割位点包含c1)；
    优选地，所述核糖核酸酶H包含核糖核酸酶HⅠ、核糖核酸酶HII中至少一种。
  5. 根据权利要求1～4任一项所述的RT-PCR引物，其特征在于：
    所述上游引物的5’端链包含多个可以被特异性切割剂切割的切割位点，例如：可以是切割位点的个数大于等于n个；或者是n～N个；或者是3～6个；其中，所述n＝N/10，N为第一序列的核苷酸个数；
    优选地，所述下游引物的5’端链包含多个可以被特异性切割剂切割的切割位点，例如：可以是切割位点的个数大于等于n个；或者是n～N个；或者是3～6个；其中，所述n＝N/10个，N为第三序列的核苷酸个数；
    优选地，所述上游引物的5’端链包含第一序列；
    优选地，所述下游引物的5’端链包含第三序列；
    优选地，所述第一序列的核苷酸个数为20～60；
    优选地，所述第三序列的核苷酸个数为20～60；
    优选地，所述第二序列的核苷酸个数为5～65；
    优选地，所述第四序列的核苷酸个数为5～65；
    优选地，所述多个可以被特异性切割剂切割的切割位点在所述上游引物的5’端链中随机分布；
    优选地，所述多个可以被特异性切割剂切割的切割位点在所述下游引物的5’端链中随机分布；
    优选地，所述第一序列与第二序列直接或间接连接；
    优选地，所述第一序列与第二序列通过以下任意一种方式连接：碱基连接、linker连接(比如：间臂(spacer3、spacer6、spacer9、spacer18等))；
    优选地，所述第三序列与第四序列直接或间接连接；
    优选地，所述第三序列与第四序列通过以下任意一种方式连接：碱基连接、linker连接(比如：间臂(spacer3、spacer6、spacer9、spacer18等))。
  6. 根据权利要求1～4任一项所述的RT-PCR引物，其特征在于：
    所述上游特异性序列和下游特异性序列分别与目标区域的两个区段相同或互补；
    优选地，所述RT-PCR引物包含多对RT-PCR引物；
    优选地，所述上游引物和下游引物中所述的特异性切割剂可以相同或不同；
    优选地，所述上游引物中的多个特异性切割剂可以相同或不同；
    优选地，所述下游引物中的多个特异性切割剂可以相同或不同；
    优选地，所述上游引物和下游引物中所述的切割位点可以相同或不同；
    优选地，所述上游引物中的多个切割位点可以相同或不同；
    优选地，所述下游引物中的多个切割位点可以相同或不同。
  7. 一种试剂盒，包含权利要求1～6任一项所述的RT-PCR引物；
    所述试剂盒还包含：权利要求1～6任一项中所述特异性切割剂；
    优选地，所述试剂盒还包含：具有3’-5’核酸外切酶活性的DNA聚合酶；
    优选地，所述试剂盒还包含：逆转录酶；
    优选地，所述试剂盒还包含：逆转录引物。
  8. 根据权利要求7所述的试剂盒，其特征在于：
    所述试剂盒包含：核酸提取试剂组合；
    优选地，所述核酸提取试剂组合是用于选自以下任意一种方法的核酸提取试剂组合：碱裂解法、酚氯仿抽提法、螯合树脂法、离心柱膜吸附法以及磁珠法；
    优选地，所述试剂盒用于d1)～d3)中任一种：
    d1)扩增目标区域；
    d2)检测目标区域；
    d3)构建靶向文库。
  9. 根据权利要求8所述的试剂盒，其特征在于：
    所述试剂盒用于检测目标区域时，所述试剂盒还包含：检测探针、核酸染料中的至少一种；
    优选地，所述检测探针包含TaqMan探针、分子信标、双杂交探针、复合探针中的至少一种；
    优选地，所述核酸染料包含EtBr、SYBR Green、SYBR Gold、GelRed、GelGreen中的至少一种。
  10. 一种多重RT-PCR扩增方法，其特征在于：利用权利要求1～6任一项所述的RT-PCR引物对待测样本的RNA进行多重RT-PCR反应；
    所述反应进一步包括：
    用所述特异性切割剂处理所述多重RT-PCR反应的体系；
    优选地，所述多重RT-PCR反应的逆转录反应与扩增反应在同一体系中进行；
    优选地，所述第一序列和第二序列为g2)、和/或所述第三序列和第四序列为h2)时，所述多重RT-PCR反应还包含所述上游引物和/或所述下游引物延伸的步骤；
    优选地，所述上游引物和/或所述下游引物延伸的步骤与所述多重RT-PCR反应的逆转录反应同时进行。
  11. 根据权利要求10所述的方法，其特征在于：
    用所述特异性切割剂处理所述多重RT-PCR的反应体系的方法可以是：
    使所述多重RT-PCR反应的体系包含所述特异性切割剂；
    优选地，用所述特异性切割剂处理所述多重RT-PCR反应的体系的步骤在所述多重RT-PCR反应的RT反应过程中或所述多重RT-PCR反应的RT反应后进行。
  12. 根据权利要求10所述的多重RT-PCR扩增方法，其特征在于：
    所述多重RT-PCR反应的体系还包含：具有3’-5’核酸外切酶活性的DNA聚合酶；
    优选地，所述多重RT-PCR反应的体系还包含：逆转录酶；
    优选地，所述多重RT-PCR反应的体系还包含：逆转录引物；
    优选地，所述RNA通过所述待测样本发生裂解反应得到；
    优选地，所述裂解反应在所述多重RT-PCR反应之前；
    优选地，所述裂解反应和所述多重RT-PCR反应在同一体系中进行；
    优选地，所述同一体系具体为同一反应容器；
    优选地，所述裂解反应后和所述多重RT-PCR反应前还包含：提取纯化步骤；
    优选地，所述提取纯化进一步包含：沉淀核酸或吸附核酸；
    优选地，所述提取纯化后还包含：洗脱或溶解核酸；
    优选地，所述裂解的方法包含：物理方式、化学方式、生物方式中的至少一种。
  13. 根据权利要求10～12任一项所述的多重RT-PCR扩增方法，其特征在于：
    所述多重RT-PCR反应的体系中还包含检测探针、核酸染料中的至少一种；
    优选地，所述检测探针包含TaqMan探针、分子信标、双杂交探针、复合探针中的至少一种；
    优选地，所述核酸染料包含EtBr、SYBR Green、SYBR Gold、GelRed、GelGreen中的至少一种。
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