Multiplex RT-PCR amplification method and use thereof

Abstract

Provided in the present invention is a multiplex RT-PCR amplification method. Primers are designed to be completely or partially complementary hairpin structures, so that the 3'-ends of the primers lose the binding and extension capabilities during an RT process; in addition, the 5'-end strands of the primers comprise one or more cleavage sites at which the 5'-end strands are cleaved by a specific cleavage agent, so that during the RT process, the 5'-end strands of the primers are cleaved at the cleavage sites by means of the specific cleavage agent, but the complementary double strands are maintained. During a high-temperature process of PCR, the cleaved 5'-end strands are free so as to release 3'-end strands to serve as primers for target amplification, thereby regaining the capability of binding primers to templates and the extension capability of 3'-ends.

Description

A multiplex RT-PCR amplification method and its application Technical Field

[0001] The invention belongs to the technical field of multiplex RT-PCR amplification, and particularly relates to a multiplex RT-PCR amplification method and application thereof. Background Art

[0002] In recent years, molecular detection technologies have rapidly developed and are widely used in fields including tumors, genetic diseases, and pathogenic microorganism detection. With the discovery of biomarkers for various diseases, an increasing number of biomarkers are being used for diagnosis, treatment, and prognosis. For example, ALK-EML gene fusion can be used for targeted drug therapy in non-small cell lung cancer, microRNA and ctDNA can be used for early screening and diagnosis of tumors, and hsp65 sequences can be used for the identification and typing of Mycobacterium tuberculosis.

[0003] Currently, the most commonly used molecular detection technology is RT-qPCR, which is used clinically due to its advantages such as simple operation and detection blocking. However, the limited number of targets that RT-qPCR can detect at a time limits its application. The invention of high-resolution melting curves has made it possible to detect multiple targets in a single tube using RT-qPCR, greatly expanding its application range. However, it is not easy to perform amplification reactions of multiplex RT-qPCR. The biggest difficulty is that the RT step and the PCR step are performed in one reaction. The by-products in the RT process will seriously affect the subsequent PCR amplification, mainly manifested in: 1) Specific primers are prone to non-specific binding with the template during the RT process, producing non-specific products, and exponentially accumulating in the subsequent PCR amplification. That is, in the RT reaction of multi-target RT-qPCR, there will be local matching between the 3 ends of different specific primers and the template. The RT enzyme will undergo an extension reaction under the condition of local matching, resulting in the production of non-specific products and exponential amplification in the subsequent PCR reaction; 2) Specific primers are prone to bind to each other and extend during the RT process, forming dimers, which accumulate exponentially in the subsequent PCR process, leading to premature termination of the entire reaction. That is, in the RT reaction of multi-target RT-qPCR, there will be local matching between the 3 ends of different specific primers. The reverse transcriptase will undergo an extension reaction under the condition of local matching, producing dimers, which exponentially amplify in the subsequent PCR process. Therefore, developing a multi-target RT-qPCR kit often requires extensive primer design, adjustment, and optimization, a tedious and complex process that increases exponentially with the number of targets. Consequently, a multiplex RT-PCR amplification method that effectively avoids nonspecific amplification and dimer formation during the RT reaction is urgently needed.

[0004] Summary of the Invention

[0005] The first aspect of the present invention aims to provide RT-PCR primers.

[0006] The second aspect of the present invention aims to provide a kit.

[0007] The third aspect of the present invention aims to provide a multiplex RT-PCR amplification method.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides RT-PCR primers, comprising: an upstream primer and a downstream primer;

[0010] The upstream primer comprises, from the 5' end to the 3' end, a first sequence and a second sequence:

[0011] g1) the first sequence is a sequence completely complementary to the upstream specific sequence, and 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; or

[0012] g2) the first sequence is a sequence that is completely complementary to the upstream specific sequence, and the second sequence is partially complementary to the first sequence (i.e., the second sequence is a partial sequence of the upstream specific sequence (preferably a 5' end sequence of the upstream specific sequence)), i.e., the upstream primer has a hairpin structure with a partially complementary 3' end (i.e., a hairpin structure with a 5' overhang); or

[0013] g3) the second sequence is an upstream specific sequence, and 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 of the upstream specific sequence)), i.e., the upstream primer has a hairpin structure with a partially complementary 5' end (i.e., a hairpin structure with a 3' overhang);

[0014] The downstream primer comprises, from the 5' end to the 3' end, a third sequence and a fourth sequence:

[0015] h1) the third sequence is a sequence completely complementary to the downstream specific sequence, and 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; or

[0016] h2) the third sequence is a sequence that is completely complementary to the downstream specific sequence, and 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 a 5' end sequence of the downstream specific sequence)), i.e., the downstream primer presents a hairpin structure with a partially complementary 3' end (i.e., a hairpin structure with a 5' overhang); or

[0017] h3) the fourth sequence is a downstream-specific sequence, and 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 of the downstream-specific sequence)), i.e., the downstream primer has a hairpin structure with a partially complementary 5' end (i.e., a hairpin structure with a 3' overhang);

[0018] The 5' end chain of the upstream primer contains one or more cleavage sites that can be cleaved by a specific cleavage agent;

[0019] The 5' end chain of the downstream primer contains one or more cleavage sites that can be cleaved by a specific cleavage agent.

[0020] Preferably, the specific cleavage agent comprises an enzymatic cleavage agent.

[0021] Preferably, 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), 3-methyladenine DNA glycosylase, 3-methyladenine DNA glycosylase II, pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase, and a thymine mismatch-DNA glycosylase (e.g., hypoxanthine-DNA glycosylase, uracil DNA glycosylase (UDG), 5-hydroxymethyluracil DNA glycosylase (HmUDG), 5-hydroxymethylcytosine DNA glycosylase, and 1,N6-vinylidene adenine DNA glycosylase).

[0022] Preferably, the cleavage site comprises at least one of c1) to c2):

[0023] c1) Ribonucleotides (rNTPs, e.g., rATP, rGTP, tCTP, rUTP);

[0024] c2) Uracil deoxyribonucleotide (dUTP).

[0025] Further preferably, the cleavage site comprises c1).

[0026] Further preferably, the enzymatic cleavage agent comprises at least one of a ribonuclease (e.g., ribonuclease H), a glycosylase (e.g., uracil-DNA glycosylase (UDG), and a USER enzyme (e.g., a mixture of uracil-DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII); and further comprises a ribonuclease (e.g., ribonuclease H).

[0027] Preferably, the ribonuclease H comprises at least one of ribonuclease HI and ribonuclease HII.

[0028] Preferably, the 5' end chain of the upstream primer contains multiple cleavage sites that can be cleaved by a specific cleavage agent, and the number of cleavage sites is greater than N / 10 bases, for example: the number of cleavage sites can be greater than or equal to (preferably greater than) n; or n to N; wherein the above n = N / 10, N is the number of nucleotides in the first sequence; or 3 to 6.

[0029] Preferably, the 5' end chain of the downstream primer contains multiple cleavage sites that can be cleaved by a specific cleavage agent, and the number of cleavage sites is greater than N / 10 bases, for example: the number of cleavage sites can be greater than or equal to (preferably greater than) n; or n to N; wherein the above n = N / 10, N is the number of nucleotides in the third sequence; or 3 to 6.

[0030] Preferably, the number of cleavage sites that can be cleaved by a specific cleavage agent is an integer.

[0031] Preferably, 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.

[0032] Preferably, 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.

[0033] Preferably, the first sequence is directly or indirectly linked to the second sequence.

[0034] Preferably, 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.)).

[0035] Preferably, the third sequence is directly or indirectly linked to the fourth sequence.

[0036] Preferably, 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.)).

[0037] Preferably, the 5' end strand of the upstream primer comprises a first sequence.

[0038] Preferably, the 5' end strand of the downstream primer comprises a third sequence.

[0039] Preferably, the number of nucleotides in the first sequence is 20-60.

[0040] Preferably, the third sequence has 20 to 60 nucleotides.

[0041] Preferably, the second sequence has 5 to 65 nucleotides.

[0042] Preferably, the fourth sequence has 5 to 65 nucleotides.

[0043] Preferably, when the upstream primer is in a hairpin structure with a complementary 3' end, the difference in the number of nucleotides between the first sequence and the second sequence is 1 to 55.

[0044] Preferably, when the upstream primer is in a hairpin structure with a complementary 5' end, the difference in the number of nucleotides between the second sequence and the first sequence is 3 to 10.

[0045] Preferably, when the downstream primer presents a hairpin structure with partial complementarity at the 3' end, the difference in the number of nucleotides between the third sequence and the fourth sequence is 1-55.

[0046] Preferably, when the downstream primer is in a hairpin structure with a complementary 5' end, the difference in the number of nucleotides between the fourth sequence and the third sequence is 3 to 10.

[0047] Preferably, the cleavage site comprises a nucleotide that can be cleaved by a specific cleavage agent.

[0048] Preferably, the specific cleavage agents in the upstream primer and the downstream primer may be the same or different.

[0049] Preferably, the multiple specific cleavage agents in the upstream primer may be the same or different.

[0050] Preferably, the multiple specific cleavage agents in the downstream primer may be the same or different.

[0051] Preferably, the cleavage sites in the upstream primer and the downstream primer may be the same or different.

[0052] Preferably, the multiple cleavage sites in the upstream primer may be the same or different.

[0053] Preferably, the multiple cleavage sites in the downstream primer may be the same or different.

[0054] Preferably, the upstream specific sequence and the downstream specific sequence are respectively identical to or complementary to the two segments of the target region.

[0055] Preferably, the RT-PCR primers comprise multiple pairs of RT-PCR primers.

[0056] The second aspect of the present invention provides a kit comprising: the RT-PCR primers according to the first aspect of the present invention.

[0057] Preferably, the kit further comprises: the specific cleavage agent described in the first aspect of the present invention.

[0058] Preferably, the kit further comprises: a DNA polymerase having 3'-5' exonuclease activity (eg, T4 DNA polymerase, DNA polymerase I (Klenow) large fragment, DNA polymerase I (E. coli)).

[0059] Preferably, the kit further comprises: reverse transcriptase.

[0060] Preferably, the kit further comprises: a reverse transcription primer.

[0061] Preferably, the reverse transcription primer is a random primer of 6 to 12 nt; further, a random primer (N6).

[0062] Preferably, the reverse transcriptase comprises at least one of AMV reverse transcriptase and M-MuLV reverse transcriptase.

[0063] Preferably, the kit comprises: a nucleic acid extraction reagent combination.

[0064] Preferably, 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.

[0065] Preferably, 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.

[0066] Preferably, the nucleic acid adsorbent comprises at least one of magnetic beads and adsorption membranes.

[0067] Preferably, the kit is used for any one of d1) to d3):

[0068] d1) amplifying the target region;

[0069] d2) detecting the target area;

[0070] d3) Constructing a library of the target region (constructing a targeted library).

[0071] Preferably, when the kit is used to detect a target area, the kit further comprises: at least one of a detection probe and a nucleic acid dye.

[0072] Preferably, the detection probe comprises at least one of a TaqMan probe, a molecular beacon, a double hybridization probe, and a composite probe; further being a TaqMan probe.

[0073] Preferably, the nucleic acid dye comprises at least one of EtBr, SYBR Green, SYBR Gold, GelRed, and GelGreen.

[0074] 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.

[0075] Preferably, the multiplex RT-PCR amplification method comprises the following steps: performing a multiplex RT-PCR reaction on the RNA of the sample to be tested using the RT-PCR primers according to the first aspect of the present invention.

[0076] Preferably, the reaction further comprises:

[0077] The multiplex RT-PCR reaction system is treated with the specific cleavage agent.

[0078] Preferably, the reverse transcription (RT) reaction and amplification (PCR) reaction of the multiplex RT-PCR reaction are performed in the same system.

[0079] Preferably, 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.

[0080] Preferably, when the first sequence and the second sequence are g2), and / or the third sequence and the fourth sequence are h2), 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.

[0081] Preferably, 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.

[0082] Preferably, the method of treating the multiplex RT-PCR reaction system with the specific cleavage agent can be:

[0083] The multiplex RT-PCR reaction system includes the specific cleavage agent.

[0084] Preferably, 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.

[0085] Preferably, 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).

[0086] Preferably, the multiplex RT-PCR reaction system further comprises: reverse transcriptase.

[0087] Preferably, the multiplex RT-PCR reaction system further comprises: reverse transcription primers.

[0088] Preferably, the RNA is obtained by a cleavage reaction of the sample to be tested.

[0089] Preferably, the cleavage reaction precedes the multiplex RT-PCR reaction.

[0090] Preferably, the cleavage reaction and the multiplex RT-PCR reaction are performed in the same system.

[0091] Preferably, the same system is specifically the same reaction container.

[0092] Preferably, the extraction and purification step is not included after the cleavage reaction and before the multiplex RT-PCR reaction.

[0093] Preferably, after the cleavage reaction and before the multiplex RT-PCR reaction, an extraction and purification step is further included.

[0094] Preferably, the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid.

[0095] Preferably, the extraction and purification further comprises: eluting or dissolving nucleic acids.

[0096] Preferably, the cleavage reaction and the multiplex RT-PCR reaction are performed in different systems, ie, not in the same system.

[0097] Preferably, the same system is specifically the same reaction container.

[0098] Preferably, the cleavage reaction further comprises an extraction and purification step.

[0099] Preferably, the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid.

[0100] Preferably, the extraction and purification further comprises: eluting or dissolving nucleic acids.

[0101] Preferably, the lysis method comprises at least one of a physical method, a chemical method, and a biological method.

[0102] Preferably, the multiplex RT-PCR reaction system further comprises at least one of a detection probe and a nucleic acid dye.

[0103] Preferably, the detection probe comprises at least one of a TaqMan probe, a molecular beacon, a double hybridization probe, and a composite probe;

[0104] Preferably, the nucleic acid dye comprises at least one of EtBr, SYBR Green, SYBR Gold, GelRed, and GelGreen.

[0105] The beneficial effects of the present invention are:

[0106] The present invention provides RT-PCR primers. The inventors creatively design primers (upstream primers and / or downstream primers) into fully 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), thereby causing the 3' end of the primer to lose its binding ability (no non-specific binding with the template and / or avoid 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 partially complementary hairpin structure also contains the primer in the reverse transcriptase and / or DNA). A step in which the 5' end of the primer is extended under the action of a reverse transcriptase to obtain a completely complementary hairpin structure), but the complementary double-stranded state is maintained during the RT process; in the subsequent high-temperature PCR process (such as thermal denaturation), the cleaved 5' end chain is freed, releasing the 3' end chain as a primer for amplifying the target region, and regaining the ability of the 3' end of the primer to bind to and extend the template; the use of the RT-PCR primer of the present invention can achieve one-step amplification and can effectively avoid the formation of dimers by multiple pairs of primers under the action of the reverse transcriptase system during the RT process, as well as the non-specific binding of multiple pairs of primers to the template under the action of the reverse transcriptase system to produce non-specific products, thereby greatly improving the efficiency of PCR amplification and the sensitivity and specificity of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] FIG1 is a schematic structural diagram of a primer of the present invention.

[0108] FIG2 is a schematic diagram showing the principle of RT-PCR amplification using the primers of the present invention.

[0109] FIG3 is a graph showing the fluorescence detection results of Example 1 and Comparative Example 1.

[0110] FIG4 is an electrophoretogram of the amplified products of Example 1 and Comparative Example 1. DETAILED DESCRIPTION

[0111] In the description of the present invention, the terms "first," "second," "third," and "fourth" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first," "second," or "third" may explicitly or implicitly include at least one of such features. In the description of the present invention, "plurality" means at least two, for example, two, three, etc., unless otherwise specifically defined.

[0112] The first aspect of the present invention provides RT-PCR primers, comprising: an upstream primer and a downstream primer;

[0113] The upstream primer comprises, from the 5' end to the 3' end, a first sequence and a second sequence:

[0114] g1) the first sequence is a sequence completely complementary to the upstream specific sequence, and 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; or

[0115] g2) the first sequence is a sequence that is completely complementary to the upstream specific sequence, and the second sequence is partially complementary to the first sequence (i.e., the second sequence is a partial sequence of the upstream specific sequence (preferably a 5' end sequence of the upstream specific sequence)), i.e., the upstream primer has a hairpin structure with a partially complementary 3' end (i.e., a hairpin structure with a 5' overhang); or

[0116] g3) the second sequence is an upstream specific sequence, and 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 of the upstream specific sequence)), i.e., the upstream primer has a hairpin structure with a partially complementary 5' end (i.e., a hairpin structure with a 3' overhang);

[0117] The downstream primer comprises, from the 5' end to the 3' end, a third sequence and a fourth sequence:

[0118] h1) the third sequence is a sequence completely complementary to the downstream specific sequence, and 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; or

[0119] h2) the third sequence is a sequence that is completely complementary to the downstream specific sequence, and 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 a 5' end sequence of the downstream specific sequence)), i.e., the downstream primer presents a hairpin structure with a partially complementary 3' end (i.e., a hairpin structure with a 5' overhang); or

[0120] h3) the fourth sequence is a downstream-specific sequence, and 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 of the downstream-specific sequence)), i.e., the downstream primer has a hairpin structure with a partially complementary 5' end (i.e., a hairpin structure with a 3' overhang);

[0121] The 5' end chain of the upstream primer contains one or more cleavage sites that can be cleaved by a specific cleavage agent;

[0122] The 5' end chain of the downstream primer contains one or more cleavage sites that can be cleaved by a specific cleavage agent.

[0123] For the first sequence, second sequence, third sequence and fourth sequence in the above RT-PCR primers, there are the following nine combinations:

[0124] a1): g1) and h1); a2): g1) and h2); a3): g1) and h3); a4): g2) and h1); a5): g2) and h2); a6): g2) and h3); a7): g3) and h1); a8): g3) and h2); a9): g3) and h3).

[0125] Preferably, 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, and the fourth sequence is completely complementary to the third sequence (i.e., the fourth sequence is the downstream specific sequence).

[0126] In the multiplex RT-PCR process, the byproducts of the RT process seriously affect the PCR process, mainly manifested in the following aspects: b1) there will be local matches between the 3' ends of different specific primers and the template, and the reverse transcriptase will undergo an extension reaction under these local matches, resulting in the production of non-specific products, which will be exponentially amplified in the subsequent PCR reaction; b2) there will be local matches between the 3' ends of different specific primers, and the reverse transcriptase will undergo an extension reaction under these local matches, producing dimers, which will be exponentially amplified in the subsequent PCR process;

[0127] In the present invention, the inventors creatively designed primers (upstream primers and / or downstream primers) to be fully 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 its binding ability (does not undergo 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 (Figure 1), so that the cleavage site of the 5' end chain of the primer is blocked during the RT process of RT-PCR. The specific cleavage agent cleaves the primer (for primers with partially complementary hairpin structures at the 3' end, this also includes the step of extending the primer under the action of reverse transcriptase and / or DNA polymerase to obtain a fully complementary hairpin primer), but maintains the complementary double-stranded state during the RT process; during the subsequent high-temperature PCR process (such as heat denaturation), the cleaved 5' end strand is freed, releasing the 3' end strand as a primer for amplification of the target region, and regaining the ability of the primer 3' end to bind to the template and extend. The amplification principle for primers with fully complementary or partially complementary hairpin structures (Figure 2) is as follows:

[0128] e1) The primer structure is a fully complementary hairpin structure: a specific sequence (an upstream specific sequence and / or a downstream specific sequence, corresponding to the second and fourth sequences described above) and a fully complementary oligonucleotide chain (e.g., a DNA chain, corresponding to the first and third sequences described above) form a "closed" hairpin structure, which can prevent non-specific binding of the primer to the template and binding between primers during the RT process. 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. During the subsequent high-temperature PCR process (e.g., thermal denaturation), the cleaved 5' end chain is freed, releasing the 3' end chain as a primer for amplifying the target region.

[0129] e2) The primer structure is a partially complementary hairpin structure:

[0130] e21) When 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 first and third sequences mentioned above) and the partial specific sequence (the 5' end sequence of the specific sequence, corresponding to the second and fourth sequences mentioned above) form a hairpin structure with partial complementarity at the 3' end (i.e., a hairpin structure with a 5' overhang), wherein the 5' end chain of the primer contains one or more cleavage sites that can be cleaved by a specific cleavage agent; first, during the RT process, the primer is extended under the action of a reverse transcriptase and / or a DNA polymerase to obtain a primer containing the entire sequence of the specific sequence and form a completely complementary hairpin structure; furthermore, the cleavage site of the 5' end chain of the primer is cleaved by the specific cleavage agent, but the complementary double-stranded state is maintained during the RT process; during the subsequent high-temperature PCR process (e.g., thermal denaturation), the cleaved 5' end chain is freed, releasing the 3' end chain as a primer for amplifying the target region;

[0131] e22) When the primer structure is a hairpin structure with partial complementarity at the 5' end, the specific sequence (upstream specific sequence and / or downstream specific sequence, corresponding to the second and fourth sequences mentioned above) and 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 first and third sequences mentioned above) form a hairpin structure with partial complementarity at the 5' end, which can prevent the primer from non-specifically binding to the template and binding between primers during the RT process. In particular, the 3' end of the primer protruding by 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, and 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 5' end chain of the primer is cut by the specific cleavage agent during the RT process of RT-PCR, but maintains a partially complementary or fully complementary double-stranded state during the RT process; in the subsequent high-temperature process of PCR (such as thermal denaturation), the cut 5' end chain is freed, releasing the 3' end chain as a primer for amplification of the target region.

[0132] The RT-PCR primers of the present invention can achieve one-step amplification and can effectively avoid the formation of dimers by multiple pairs of primers under the action of the reverse transcriptase system during the RT process, as well as the non-specific binding of multiple pairs of primers to the template under the action of the reverse transcriptase system to produce non-specific products, thereby greatly improving the efficiency of PCR amplification and the sensitivity and specificity of detection.

[0133] Preferably, the upstream specific sequence and the downstream specific sequence are respectively identical to or complementary to the two segments of the target region.

[0134] Preferably, the design of the specific sequences (upstream specific sequence and / or downstream specific sequence) in the primers (upstream primer and / or downstream primer) 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 the same as 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 the same as the negative chain (i.e., antisense chain, cDNA second chain) sequence.

[0135] Preferably, the upstream specific sequence is complementary to the upstream negative chain sequence of the target region or is identical to the positive chain sequence.

[0136] Preferably, the downstream specific sequence is complementary to the downstream positive chain sequence of the target region or is identical to the negative chain sequence.

[0137] Preferably, the specific cleavage agent comprises an enzymatic cleavage agent.

[0138] Preferably, 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), 3-methyladenine DNA glycosylase, 3-methyladenine DNA glycosylase II, pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase, and a thymine mismatch-DNA glycosylase (e.g., hypoxanthine-DNA glycosylase, uracil DNA glycosylase (UDG), 5-hydroxymethyluracil DNA glycosylase (HmUDG), 5-hydroxymethylcytosine DNA glycosylase, and 1,N6-vinylidene adenine DNA glycosylase).

[0139] Preferably, the cleavage site comprises at least one of c1) to c2):

[0140] c1) ribonucleotides (rNTPs, e.g., rATP, rGTP, tCTP, rUTP), whose corresponding specific cleavage agent is a ribonuclease (e.g., RNase H). When the rNTP is rUTP, 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);

[0141] c2) uracil deoxyribonucleotide (dUTP), the corresponding specific cleavage agent is uracil-DNA glycosylase (UDG) or USER enzyme (a mixture of uracil-DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII).

[0142] Further preferably, the cleavage site comprises c1).

[0143] Further preferably, the enzymatic cleavage agent comprises at least one of a ribonuclease (e.g., ribonuclease H), a glycosylase (e.g., uracil-DNA glycosylase (UDG), and a USER enzyme (e.g., a mixture of uracil-DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII); and further comprises a ribonuclease (e.g., ribonuclease H).

[0144] Preferably, the ribonuclease H comprises at least one of ribonuclease HI and ribonuclease HII.

[0145] Preferably, the specific cleavage agents in the upstream primer and the downstream primer may be the same or different.

[0146] Preferably, the multiple specific cleavage agents in the upstream primer may be the same or different.

[0147] Preferably, the multiple specific cleavage agents in the downstream primer may be the same or different.

[0148] Preferably, the cleavage sites in the upstream primer and the downstream primer may be the same or different.

[0149] Preferably, the multiple cleavage sites in the upstream primer may be the same or different.

[0150] Preferably, the multiple cleavage sites in the downstream primer may be the same or different.

[0151] Preferably, the 5' end chain of the upstream primer contains multiple cleavage sites that can be cleaved by a specific cleavage agent, and the number of cleavage sites is greater than N / 10 bases, for example: the number of cleavage sites can be greater than or equal to (preferably greater than) n; or n to N; wherein the above n = N / 10, N is the number of nucleotides in the first sequence; or 3 to 6.

[0152] Preferably, the 5' end chain of the downstream primer contains multiple cleavage sites that can be cleaved by a specific cleavage agent, and the number of cleavage sites is greater than N / 10 bases, for example: the number of cleavage sites can be greater than or equal to (preferably greater than) n; or n to N, wherein the above n = N / 10, N is the number of nucleotides in the third sequence; or 3 to 6.

[0153] Preferably, the number of cleavage sites that can be cleaved by a specific cleavage agent is an integer.

[0154] Preferably, 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.

[0155] Preferably, 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.

[0156] Preferably, the first sequence is directly or indirectly linked to the second sequence.

[0157] Preferably, 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.)).

[0158] Preferably, the third sequence is directly or indirectly linked to the fourth sequence.

[0159] Preferably, 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.)).

[0160] Preferably, the 5' end strand of the upstream primer comprises a first sequence.

[0161] Preferably, the 5' end strand of the downstream primer comprises a third sequence.

[0162] Preferably, the number of nucleotides in the first sequence is 20-60.

[0163] Preferably, the third sequence has 20 to 60 nucleotides.

[0164] Preferably, the second sequence has 5 to 65 nucleotides.

[0165] Preferably, the fourth sequence has 5 to 65 nucleotides.

[0166] Preferably, when the upstream primer is in a hairpin structure with a complementary 3' end, the difference in the number of nucleotides between the first sequence and the second sequence is 1 to 55.

[0167] Preferably, when the upstream primer is in a hairpin structure with a complementary 5' end, the difference in the number of nucleotides between the second sequence and the first sequence is 3 to 10.

[0168] Preferably, when the downstream primer presents a hairpin structure with partial complementarity at the 3' end, the difference in the number of nucleotides between the third sequence and the fourth sequence is 1-55.

[0169] Preferably, when the downstream primer is in a hairpin structure with a complementary 5' end, the difference in the number of nucleotides between the fourth sequence and the third sequence is 3 to 10.

[0170] Preferably, the cleavage site comprises a nucleotide that can be cleaved by a specific cleavage agent.

[0171] Preferably, the RT-PCR primer is a DNA fragment or an analog thereof.

[0172] Preferably, the RT-PCR primers comprise multiple pairs of RT-PCR primers.

[0173] Preferably, the plurality of pairs is two or more pairs.

[0174] Preferably, the RT-PCR primers for detecting the SARS-COV-2 gene comprise: an upstream primer and a downstream primer;

[0175] The 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;

[0176] The 5' end of the upstream primers with sequences as shown in SEQ ID NO. 1, 4, 7, 10, and 13 each independently comprises one or more cleavage sites that can be cleaved by a specific cleavage agent;

[0177] The 5' ends of the downstream primers with sequences as shown in SEQ ID NOs. 2, 5, 8, 11, and 14 each independently contain one or more cleavage sites that can be cleaved by a specific cleavage agent.

[0178] Preferably, the specific cleavage agent comprises an enzymatic cleavage agent.

[0179] Preferably, 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), 3-methyladenine DNA glycosylase, 3-methyladenine DNA glycosylase II, pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase, and a thymine mismatch-DNA glycosylase (e.g., hypoxanthine-DNA glycosylase, uracil DNA glycosylase (UDG), 5-hydroxymethyluracil DNA glycosylase (HmUDG), 5-hydroxymethylcytosine DNA glycosylase, and 1,N6-vinylidene adenine DNA glycosylase).

[0180] Preferably, the cleavage site comprises at least one of c1) to c2):

[0181] c1) ribonucleotides (rNTPs, e.g., rATP, rGTP, tCTP, rUTP), whose corresponding specific cleavage agent is a ribonuclease (e.g., RNase H). When the rNTP is rUTP, its corresponding specific cleavage agent may also be uracil-DNA glycosylase (UDG) or USER enzyme (e.g., a mixture of uracil-DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII);

[0182] c2) uracil deoxyribonucleotide (dUTP), the corresponding specific cleavage agent is uracil-DNA glycosylase (UDG) or USER enzyme (for example, a mixture of uracil-DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII).

[0183] Further preferably, the cleavage site comprises c1).

[0184] Further preferably, the enzymatic cleavage agent comprises at least one of a ribonuclease (e.g., ribonuclease H), a glycosylase (e.g., uracil-DNA glycosylase (UDG), and a USER enzyme (e.g., a mixture of uracil-DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII); and further comprises a ribonuclease (e.g., ribonuclease H).

[0185] Preferably, the ribonuclease H comprises at least one of ribonuclease HI and ribonuclease HII.

[0186] Preferably, the positions of the ribonucleotides of the upstream primers whose sequences are shown in SEQ ID NO. 1, 4, 7, 10, 13 are shown in Table 1 and the notes.

[0187] Preferably, the positions of the ribonucleotides of the downstream primers whose sequences are shown in SEQ ID NOs. 2, 5, 8, 11, and 14 are shown in Table 2 and the notes.

[0188] Preferably, the RT-PCR primers are used to amplify RNA of the sample to be tested.

[0189] Preferably, the sample to be tested comprises cells, tissues, body fluids, microorganisms, saliva, urine, sputum, feces, throat swabs, and nasal swabs.

[0190] Preferably, the body fluid comprises at least one of tissue fluid, lymph fluid, blood, and cerebrospinal fluid.

[0191] Preferably, the microorganism comprises at least one of bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, and spirochetes.

[0192] Preferably, the RNA comprises at least one of mRNA, lncRNA, small RNA, cfRNA and tRNA.

[0193] Preferably, the small RNA may be at least one of miRNA, siRNA and piRNA.

[0194] The second aspect of the present invention provides a kit comprising: the RT-PCR primers according to the first aspect of the present invention.

[0195] Preferably, the kit further comprises: the specific cleavage agent, which is used to cleave the cleavage site of the 5' end chain of the primer (upstream primer and / or downstream primer) of the fully complementary or partially complementary hairpin structure during the RT process of RT-PCR.

[0196] Preferably, 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). 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 second sequence is partially complementary to the first sequence and / or the fourth sequence is partially complementary to the third sequence).

[0197] Preferably, the kit further comprises: a reverse transcription reaction mixture, which is used for reverse transcription reaction (ie, reverse transcription of RNA into cDNA).

[0198] Preferably, the reverse transcription reaction mixture comprises: at least one of: reverse transcriptase, DNA polymerase, reverse transcription buffer, reverse transcription primer, dNTPs, and RNA enzyme inhibitor; preferably, the reverse transcription reaction mixture comprises: reverse transcriptase, DNA polymerase, reverse transcription buffer, dNTPs, RNA enzyme inhibitor and reverse transcription primer; the reverse transcriptase and / or 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 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 component 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.

[0199] Preferably, the reverse transcription primer is a random primer of 6 to 12 nt; further, a random primer (N6).

[0200] Preferably, the reverse transcriptase comprises at least one of AMV reverse transcriptase and M-MuLV reverse transcriptase.

[0201] Preferably, the kit further comprises: a PCR reaction mixture for PCR reaction.

[0202] Preferably, the PCR reaction mixture comprises: DNA polymerase, Mg 2+ , PCR buffer, at least one of dNTPs; further preferably, the PCR reaction mixture further comprises: DNA polymerase, Mg 2+ , PCR buffer and dNTPs; the DNA polymerase therein 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 primer structure is a hairpin structure with partial complementarity at the 3' end); for the components already contained in the kit, the PCR reaction mixture can reduce the component 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.

[0203] Preferably, the DNA polymerase is a thermostable DNA polymerase (such as Taq DNA polymerase); further, it is a Taq hot-start DNA polymerase.

[0204] Preferably, the kit comprises: a nucleic acid extraction reagent combination.

[0205] Preferably, 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.

[0206] Preferably, 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.

[0207] Preferably, the nucleic acid adsorbent comprises at least one of magnetic beads and adsorption membranes.

[0208] Preferably, the kit is used for any one of d1) to d3):

[0209] d1) amplifying the target region;

[0210] d2) detecting the target area;

[0211] d3) Constructing a library of the target region (constructing a targeted library).

[0212] Preferably, when the kit is used to detect a target area, the kit further 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.

[0213] Preferably, the detection probe comprises at least one of a TaqMan probe, a molecular beacon, a double hybridization probe, and a composite probe; further being a TaqMan probe.

[0214] Preferably, the nucleic acid dye comprises at least one of EtBr, SYBR Green, SYBR Gold, GelRed, and GelGreen.

[0215] Preferably, when the kit is used to detect the SARS-COV-2 gene, the RT-PCR primers are the RT-PCR primers for detecting the SARS-COV-2 gene according to the first aspect of the present invention.

[0216] Preferably, 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, and 15.

[0217] Preferably, the two ends of the probe for detecting the SARS-COV-2 gene are respectively connected to a quenching group and a fluorescent group.

[0218] Preferably, the fluorescent group comprises at least one of FAM, TET, VIC, HEX, Cy5, ROX, Texas Red, Tamara, and JOE; further preferably, the fluorescent group comprises FAM.

[0219] Preferably, the quencher group comprises at least one of BHQ1, BHQ2, BHQ3, and MGB; further preferably, the quencher group comprises BHQ1.

[0220] Preferably, when the kit is used to construct a library of a target region, the kit further comprises: a reagent for adding a sequencing adapter.

[0221] Preferably, 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.

[0222] The third aspect of the present invention provides a sequencing reagent kit, comprising: a reagent kit according to the second aspect of the present invention.

[0223] Preferably, the sequencing reagent set further comprises: a sequencing reagent kit.

[0224] A fourth aspect of the present invention provides a sequencing system comprising: a sequencer and the sequencing reagent set according to the third aspect of the present invention.

[0225] 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.

[0226] Preferably, the multiplex RT-PCR amplification method comprises the following steps: performing a multiplex RT-PCR reaction on the RNA of the sample to be tested using the RT-PCR primers according to the first aspect of the present invention.

[0227] Preferably, the reaction further comprises:

[0228] The multiplex RT-PCR reaction system is treated with the specific cleavage agent.

[0229] Preferably, the reverse transcription (RT) reaction and amplification (PCR) reaction of the multiplex RT-PCR reaction are performed in the same system.

[0230] Preferably, 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.

[0231] Preferably, when the first sequence and the second sequence are g2), and / or the third sequence and the fourth sequence are h2), 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.

[0232] Preferably, 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.

[0233] In the method of the present invention, the primers (upstream primers and / or downstream primers) are designed to have 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 its binding ability (does not bind to the template non-specifically 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 a specific cleavage agent during the RT process of RT-PCR (the 3' end partially complementary hairpin structure also contains primers that are cleaved by a reverse transcriptase and / or DNA polymerase). The method comprises the following steps: extending the 5' end strand under the action of the reverse transcriptase to obtain a completely complementary hairpin structure; however, maintaining the complementary double-stranded state during the RT process; in the subsequent high-temperature PCR process (e.g., thermal denaturation), the cleaved 5' end strand is freed, releasing the 3' end strand as a primer for amplifying the target region, and regaining the ability of the 3' end primer to bind to and extend the template; thereby, the method of the present invention can achieve one-step amplification, and can effectively avoid the formation of dimers of multiple pairs of primers under the action of the reverse transcriptase system during the RT process, as well as the non-specific binding of multiple pairs of primers to the template under the action of the reverse transcriptase system to produce non-specific products, thereby greatly improving the efficiency of PCR amplification and the sensitivity and specificity of detection.

[0234] Preferably, 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.

[0235] Preferably, the method for treating the multiplex RT-PCR reaction system with the specific cleavage agent can be:

[0236] The multiplex RT-PCR reaction system includes the specific cleavage agent.

[0237] Preferably, 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), 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 the reverse transcriptase). 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 hairpin structure with partial complementarity at the 3' end).

[0238] Preferably, the multiplex RT-PCR reaction system further comprises: a reverse transcription reaction mixture, which is used for reverse transcription reaction (ie, reverse transcription of RNA into cDNA).

[0239] Preferably, the reverse transcription reaction mixture contains: at least one of: reverse transcriptase, DNA polymerase, reverse transcription buffer, reverse transcription primer, dNTPs, and RNA enzyme inhibitor; preferably, the reverse transcription reaction mixture contains: reverse transcriptase, DNA polymerase, reverse transcription buffer, dNTPs, RNA enzyme inhibitor and reverse transcription primer; the reverse transcriptase and / or 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 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 component 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.

[0240] Preferably, the reverse transcription primer is a random primer of 6 to 12 nt; further, a random primer (N6).

[0241] Preferably, the reverse transcriptase comprises at least one of AMV reverse transcriptase and M-MuLV reverse transcriptase.

[0242] Preferably, the multiplex RT-PCR reaction system further comprises: a PCR reaction mixture, which is used for PCR reaction.

[0243] Preferably, the PCR reaction mixture comprises: DNA polymerase, Mg 2+ , PCR buffer, at least one of dNTPs; further preferably, the PCR reaction mixture further comprises: DNA polymerase, Mg 2+, PCR buffer and dNTPs; the DNA polymerase therein 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 component 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.

[0244] Preferably, the DNA polymerase is a thermostable DNA polymerase (such as Taq DNA polymerase); further, it is a Taq hot-start DNA polymerase.

[0245] Preferably, 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.

[0246] Preferably, the RNA is obtained by a cleavage reaction of the sample to be tested.

[0247] Preferably, the cleavage reaction precedes the multiplex RT-PCR reaction.

[0248] Preferably, the cleavage reaction and the multiplex RT-PCR reaction are performed in the same system.

[0249] Preferably, the same system is specifically the same reaction container.

[0250] Preferably, the extraction and purification step is not included after the cleavage reaction and before the multiplex RT-PCR reaction.

[0251] Preferably, after the cleavage reaction and before the multiplex RT-PCR reaction, the process further comprises: an extraction and purification step to remove impurities such as salts and organic agents.

[0252] Preferably, the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid.

[0253] Preferably, the extraction and purification further comprises: eluting or dissolving nucleic acids.

[0254] Preferably, the cleavage reaction and the multiplex RT-PCR reaction are performed in different systems, ie, not in the same system.

[0255] Preferably, the same system is specifically the same reaction container.

[0256] Preferably, the cleavage reaction further comprises an extraction and purification step to remove impurities such as salts and organic agents.

[0257] Preferably, the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid.

[0258] Preferably, the extraction and purification further comprises: eluting or dissolving nucleic acids.

[0259] Preferably, the lysis method comprises at least one of a physical method, a chemical method, and a biological method.

[0260] Preferably, 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.

[0261] Preferably, the chemical method comprises at least one of a surfactant method (SDS method) and an alkaline lysis method.

[0262] Preferably, the biological method comprises: enzymatic method, such as cleavage by enzymes such as lysozyme and proteinase K.

[0263] 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.

[0264] Preferably, 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).

[0265] Preferably, a method for detecting the SARS-COV-2 gene comprises the following steps: performing a multiplex RT-PCR reaction on the RNA of the sample to be tested using the RT-PCR primers for detecting the SARS-COV-2 gene in the first aspect of the present invention.

[0266] Preferably, the RT-PCR reaction system further comprises the probe for detecting the SARS-COV-2 gene in the second aspect of the present invention.

[0267] Preferably, the method is a method for non-diagnostic purposes.

[0268] 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;

[0269] The method further comprises the following steps: adding a sequencing adapter to the RT-PCR product to obtain a sequencing library.

[0270] Preferably, a circularization reaction may be further included after adding sequencing adapters before obtaining the sequencing library, that is, circularizing the amplified linear library.

[0271] Preferably, the method further comprises the step of purifying the sequencing library.

[0272] Preferably, the purification is performed using magnetic beads.

[0273] 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.

[0274] Preferably, the sequencing method comprises the following steps: preparing a library; sequencing;

[0275] The method for preparing a library is the method for constructing a targeted library according to the seventh aspect of the present invention.

[0276] Preferably, the sequencing further includes the following steps: library quality inspection.

[0277] 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.

[0278] Preferably, the method for obtaining target region gene information comprises the following steps: sequencing to obtain sequencing data; obtaining target region gene information; the information comprises at least one of sequence information and variation information;

[0279] The sequencing method is the sequencing method of the eighth aspect of the present invention.

[0280] Preferably, when the information includes variation information, the method for obtaining 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.

[0281] Preferably, the variation includes at least one of gene fusion, SNV, gene deletion, and gene insertion mutation.

[0282] Preferably, the method is a method for non-diagnostic purposes.

[0283] In a tenth aspect, 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);

[0284] f1) constructing a targeted library;

[0285] f2) preparing products for constructing targeted libraries;

[0286] f3) sequencing;

[0287] f4) preparing products for sequencing;

[0288] f5) obtaining target region gene information;

[0289] f6) preparing products for obtaining genetic information of target regions;

[0290] f7) multiplex RT-PCR amplification;

[0291] f8) preparing products for multiplex RT-PCR amplification;

[0292] f9) detecting the target area;

[0293] f10) preparing a product for detecting the target area.

[0294] Preferably, the applications described in f5) and f9) are for non-diagnostic purposes.

[0295] The present invention is further described in detail below through specific examples.

[0296] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0297] The experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturers. The materials and reagents used in these examples were commercially available unless otherwise specified.

[0298] The methods of the following embodiments / comparative examples are used to detect multiple gene sequences of the new coronavirus, including 5 pairs of primers for amplifying 5 target sequences respectively to increase the probability of detection of the new coronavirus-specific sequence; the RT-qPCR reagent used is Novozymes (HiScript II One Step RT-PCR Kit, Catalog No. P611-01), the RNase HII enzyme is from NEB, Catalog No. M0288S, and the Klenow enzyme is from NEB, USA, Catalog No. M0210S.

[0299] Example 1 A method for detecting SARS-COV-2 genes using multiplex RT-qPCR

[0300] A method for detecting SARS-CoV-2 genes by multiplex RT-qPCR, comprising the following steps:

[0301] An RT-qPCR system was prepared in a PCR tube according to the reaction system shown in Table 1 (wherein, the amplification primer and probe sequences are shown in Table 2, i.e., the amplification primers can form a completely complementary hairpin structure). RT-qPCR reactions were performed on a Bio-rad PCR instrument with the following reaction program: 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 infinity.

[0302] Table 1 RT-qPCR system

[0303] Table 2 Amplification primer and probe sequences

[0304] Note: The above primers / probes were mixed at a concentration of 20 μM for each primer / probe to obtain a primer / probe pool with a total concentration of 20 μM; the underlined bases are ribonucleic acid bases, i.e., the corresponding nucleotides are ribonucleotides (rNTPs); FAM is a fluorescent group, and BHQ1 is a quenching group; the bold parts in the above primers are specific sequences (upstream specific sequence or downstream specific sequence).

[0305] Comparative Example 1 A method for detecting SARS-COV-2 genes using multiplex RT-qPCR

[0306] A method for detecting SARS-CoV-2 genes by multiplex RT-qPCR, comprising the following steps:

[0307] The RT-qPCR system was prepared in a PCR tube according to the reaction system shown in Table 3 (wherein the amplification primer and probe sequences are shown in Table 4). The RT-qPCR reaction was performed on a Bio-rad PCR instrument with the following reaction program: 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 ∞.

[0308] Table 3 RT-qPCR system

[0309] Table 4 Amplification primer and probe sequences

[0310] Note: The above primers / probes were mixed at a concentration of 20 μM each to obtain a primer / probe pool with a total concentration of 20 μM; FAM is the fluorescent group and BHQ1 is the quenching group.

[0311] Effect embodiment

[0312] The methods of 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.

[0313] 1. Fluorescence detection results

[0314] The fluorescence detection results using the methods of Example 1 and Comparative Example 1 are shown in Table 5 and Figure 3. When the concentrations of the standard are 100 and 1000 copies / ml, the CT value (Cycle Threshold Value) of the method of Example 1 is lower than that of Comparative Example 1. When the concentration of the standard is 10 copies / ml, the CT value of the method of Example 1 is 39.3, while the method of Comparative Example 1 cannot detect it. It can be seen that compared with the method of Comparative Example 1, the method of Example 1 can significantly improve the efficiency of PCR amplification and the sensitivity of detection.

[0315] Table 5 Fluorescence detection results

[0316] 2. Electrophoresis test results

[0317] The products obtained by RT-qPCR in Example 1 and Comparative Example 1 were subjected to electrophoresis, and the results are shown in Figure 4: Compared with the product obtained by the method of Comparative Example 1, the product obtained by the method of Example 1 is more uniform (especially when the concentration of the standard is low, that is, when the viral load is low), it can be seen that the method of Example 1 is more specific.

[0318] When 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 each reduced by 3 nucleotides), they have the same effect as Example 1.

[0319] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. RT-PCR primers, including: upstream primer and downstream primer; The upstream primer comprises, from the 5' end to the 3' end, a first sequence and a second sequence: g1) the first sequence is a sequence that is completely complementary to an upstream specific sequence, and the second sequence is completely complementary to the first sequence; or g2) the first sequence is a sequence that is completely complementary to the upstream specific sequence, and the second sequence is partially complementary to the first sequence; or g3) the second sequence is an upstream specific sequence, and the first sequence is partially complementary to the second sequence; The downstream primer comprises, from the 5' end to the 3' end, a third sequence and a fourth sequence: h1) the third sequence is a sequence that is completely complementary to the downstream specific sequence, and the fourth sequence is completely complementary to the third sequence; or h2) the third sequence is a sequence that is completely complementary to the downstream specific sequence, and the fourth sequence is partially complementary to the third sequence; or h3) the fourth sequence is a downstream specific sequence, and the third sequence is partially complementary to the fourth sequence; The 5' end strand 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.

2. The RT-PCR primer according to claim 1, characterized in that: The specific cleavage agent comprises an enzymatic cleavage agent; Preferably, 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), 3-methyladenine DNA glycosylase, 3-methyladenine DNA glycosylase II, pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase, and a thymine mismatch-DNA glycosylase (e.g., hypoxanthine-DNA glycosylase, uracil DNA glycosylase (UDG), 5-hydroxymethyluracil DNA glycosylase (HmUDG), 5-hydroxymethylcytosine DNA glycosylase, and 1,N6-vinylidene adenine DNA glycosylase)).

3. The RT-PCR primer according to claim 2, characterized in that: The cleavage site comprises at least one of c1) to c2): c1) ribonucleotides; c2) Uracil deoxyribonucleotide.

4. The RT-PCR primer according to claim 3, characterized in that: The enzymatic cleavage agent comprises at least one of a ribonuclease (e.g., ribonuclease H), a glycosylase (e.g., uracil-DNA glycosylase (UDG), and a USER enzyme (e.g., a mixture of uracil-DNA glycosylase (UDG) and DNA glycosylase-lyase Endo VIII)); and further comprises a ribonuclease; Preferably, the cleavage site comprises c1); Preferably, the ribonuclease H comprises at least one of ribonuclease HI and ribonuclease HII.

5. The RT-PCR primer according to any one of claims 1 to 4, characterized in that: The 5' end chain of the upstream primer contains a plurality of cleavage sites that can be cleaved by a specific cleavage agent, for example: the number of cleavage sites may be greater than or equal to n; or n to N; or 3 to 6; wherein n = N / 10, N is the number of nucleotides in the first sequence; Preferably, the 5' end chain of the downstream primer contains multiple cleavage sites that can be cleaved by a specific cleavage agent, for example: the number of cleavage sites may be greater than or equal to n; or n to N; or 3 to 6; wherein n = N / 10, N is the number of nucleotides in the third sequence; Preferably, the 5' end strand of the upstream primer comprises a first sequence; Preferably, the 5' end strand of the downstream primer comprises a third sequence; Preferably, the number of nucleotides in the first sequence is 20 to 60; Preferably, the number of nucleotides in the third sequence is 20 to 60; Preferably, the number of nucleotides in the second sequence is 5 to 65; Preferably, the number of nucleotides in the fourth sequence is 5 to 65; Preferably, 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; Preferably, 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; Preferably, the first sequence is directly or indirectly linked to the second sequence; Preferably, the first sequence and the second sequence are connected by any of the following methods: base connection, linker connection (for example: spacer (spacer3, spacer6, spacer9, spacer18, etc.)); Preferably, the third sequence is directly or indirectly connected to the fourth sequence; Preferably, 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.)).

6. The RT-PCR primer according to any one of claims 1 to 4, characterized in that: The upstream specific sequence and the downstream specific sequence are respectively identical to or complementary to the two segments of the target region; Preferably, the RT-PCR primers comprise multiple pairs of RT-PCR primers; Preferably, the specific cleavage agents in the upstream primer and the downstream primer may be the same or different; Preferably, the multiple specific cleavage agents in the upstream primer may be the same or different; Preferably, the multiple specific cleavage agents in the downstream primer may be the same or different; Preferably, the cleavage sites in the upstream primer and the downstream primer may be the same or different; Preferably, the multiple cleavage sites in the upstream primer may be the same or different; Preferably, the multiple cleavage sites in the downstream primer may be the same or different.

7. A kit comprising the RT-PCR primer according to any one of claims 1 to 6; The kit further comprises: the specific cleavage agent according to any one of claims 1 to 6; Preferably, the kit further comprises: a DNA polymerase having 3'-5' exonuclease activity; Preferably, the kit further comprises: reverse transcriptase; Preferably, the kit further comprises: a reverse transcription primer.

8. The kit according to claim 7, characterized in that: The kit comprises: a nucleic acid extraction reagent combination; Preferably, the nucleic acid extraction reagent combination is a nucleic acid extraction reagent combination selected from 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; Preferably, the kit is used for any one of d1) to d3): d1) amplifying the target region; d2) detecting the target area; d3) Construction of targeted library.

9. The kit according to claim 8, characterized in that: 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; Preferably, the detection probe comprises at least one of a TaqMan probe, a molecular beacon, a dual hybridization probe, and a composite probe; Preferably, the nucleic acid dye comprises at least one of EtBr, SYBR Green, SYBR Gold, GelRed, and GelGreen.

10. A multiplex RT-PCR amplification method, characterized in that: Using the RT-PCR primers described in any one of claims 1 to 6 to perform multiple RT-PCR reactions on the RNA of the sample to be tested; The reaction further comprises: Treating the multiplex RT-PCR reaction system with the specific cutting agent; Preferably, the reverse transcription reaction and amplification reaction of the multiplex RT-PCR reaction are carried out in the same system; Preferably, when the first sequence and the second sequence are g2), and / or the third sequence and the fourth sequence are h2), the multiplex RT-PCR reaction further comprises a step of extending the upstream primer and / or the downstream primer; Preferably, the step of extending the upstream primer and / or the downstream primer is performed simultaneously with the reverse transcription reaction of the multiplex RT-PCR reaction.

11. The method according to claim 10, characterized in that: The method of treating the multiplex RT-PCR reaction system with the specific cleavage agent may be: The multiplex RT-PCR reaction system includes the specific cutting agent; Preferably, 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.

12. The multiplex RT-PCR amplification method according to claim 10, characterized in that: The multiplex RT-PCR reaction system further comprises: a DNA polymerase having 3'-5' exonuclease activity; Preferably, the multiplex RT-PCR reaction system further comprises: reverse transcriptase; Preferably, the multiplex RT-PCR reaction system further comprises: a reverse transcription primer; Preferably, the RNA is obtained by a cleavage reaction of the sample to be tested; Preferably, the cleavage reaction precedes the multiplex RT-PCR reaction; Preferably, the cleavage reaction and the multiplex RT-PCR reaction are performed in the same system; Preferably, the same system is specifically the same reaction container; Preferably, after the cleavage reaction and before the multiplex RT-PCR reaction, the method further comprises: an extraction and purification step; Preferably, the extraction and purification further comprises: precipitating nucleic acid or adsorbing nucleic acid; Preferably, the extraction and purification further comprises: eluting or dissolving nucleic acids; Preferably, the lysis method comprises at least one of a physical method, a chemical method and a biological method.

13. The multiplex RT-PCR amplification method according to any one of claims 10 to 12, characterized in that: The multiplex RT-PCR reaction system further comprises at least one of a detection probe and a nucleic acid dye; Preferably, the detection probe comprises at least one of a TaqMan probe, a molecular beacon, a dual hybridization probe, and a composite probe; Preferably, the nucleic acid dye comprises at least one of EtBr, SYBR Green, SYBR Gold, GelRed, and GelGreen.

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