Nucleic acid isothermal amplification and detection system and detection method thereof

Abstract

Provided are a nucleic acid isothermal amplification and detection system and a detection method thereof. By means of the combined use of a nicking endonuclease, a DNA polymerase (reverse transcriptase is added for an RNA template), and an endoribonuclease, a primer containing a cleavage recognition sequence of the nicking endonuclease and a probe containing at least one ribonucleotide base are designed, and base modification is performed on the primer and the probe, so that, during isothermal amplification, the signal intensity for detecting a target nucleic acid is continuously amplified, the detection sensitivity is significantly enhanced. Additionally, the present application can also avoid mismatches to reduce non-specific amplification, improve the detection specificity, prevent missed detection and false detection, shorten detection time, and widen the application range.

Description

A nucleic acid isothermal amplification detection system and detection method thereof Technical Field

[0001] The present invention belongs to the field of gene detection, and in particular relates to a nucleic acid isothermal amplification detection system and a detection method thereof. Background Art

[0002] Nucleic acid testing has been widely used in the fields of infectious disease or genetic disease diagnosis, epidemiological surveys, and tumor mutation gene detection. Compared with immunological detection technology, molecular diagnosis has higher sensitivity and more accurate and reliable results. The most critical step in nucleic acid detection is nucleic acid amplification technology. Polymerase chain reaction (PCR) is a classic and mature in vitro nucleic acid amplification method. However, large-scale precision instruments and complex operation steps still limit its application scenarios. In recent years, constant temperature in vitro nucleic acid amplification technology has quietly emerged, such as LAMP (loop-mediated isothermal amplification technology), TMA (transcription-mediated amplification method), RPA (recombinase polymerase amplification), SDA (strand displacement amplification technology), HDA (helicase-dependent amplification technology), NASBA (nucleic acid sequence-dependent amplification technology), RCA (rolling circle replication nucleic acid amplification technology), NEAR (nickase amplification reaction technology), etc.

[0003] NEAR (nickase amplification reaction) creates a small incision on the DNA surface, allowing the polymerase to find that location and begin replication, continuously forming incisions and replicating. This is a very ingenious method that eliminates the need for PCR reactions and thermal cycling, making the nucleic acid amplification process much simpler and faster.

[0004] With the discovery of single-strand nicking restriction endonucleases (also known as nickases), people have gradually replaced double-strand restriction endonucleases and modified single nucleotide methods with them. In 2000, the scientific team of New England Biolabs, Inc. established an amplification reaction based on Nt.BstNBI nickase and Bst DNA polymerase, and proved that other nickases such as Nt.Alw I or N.Mly I can also be used for double-strand amplification (WO01 / 94544A2). Van Ness J. et al. 2003 used DNA polymerase and nickase as the amplification system for EXPAR (exponential amplification reaction) detection test. The amplification speed was extremely fast, and 10 6However, team member Eric Tan et al. (2008) discovered that nonspecific amplification or false positives could not be ruled out during amplification. They controlled and reduced the extent of nonspecific amplification by splitting the buffer and enzyme components and mixing them after heating. In 2007, Traci Kiesling et al. used Nt.Alw I and Nt.BstNBI with DNA polymerase for amplification detection reactions and named it NESA amplification technology. Jianwei Jeffery Li et al. also performed similar amplification detection using N.Bbvc IA, N.Bbvc IB, and N.BstNBI in 2008.

[0005] In the 1960s, Kornberg proposed that DNA polymerase has the possibility of DNA synthesis ability that is independent of primer or template. As research continues to deepen, this ability of DNA polymerase is confirmed, and is referred to as Ab initio, initial synthesis (1997,1998, Ogata etc.). And in 2006, Xingguo Liang etc. found that, when restriction endonuclease or nickase exist simultaneously, effectively promote this non-template or primer-dependent synthesis. Subsequently, Xingguo Liang research team, Russian research team Nadezhda V.Zyrina etc. studied the amplification model of initial synthesis respectively, confirmed that under the existence of nickase or restriction endonuclease, DNA polymerase can synthesize short nickase or restriction endonuclease recognition sequence or close repeat tandem fragment, then these fragments are rapidly amplified and extended, form a large amount of non-specific Ab initio amplification products, become one of the important factors that limit the utilization of this type of enzyme to carry out nucleic acid amplification.

[0006] Ribonuclease H (RNase H) is a functional enzyme widely found in prokaryotes and eukaryotes. It participates in the excision repair process of genomic sequences and effectively degrades RNA strands in RNA-DNA hybrids, making it a commonly used tool enzyme. In 1973, Miller et al. first discovered RNase H from Escherichia coli, and RNase H was subsequently cloned and mapped. In 1990, Itaya et al. successfully demonstrated the presence of a second RNase H (RNase H2) in Escherichia coli K-12, which also degrades RNA in RNA-DNA hybrids. In 1999, Ohtani et al. identified a third RNase H (RNase H3) from Bacillus subtilis.

[0007] The inventors previously developed a unique isothermal amplification detection technology (CN202211195457). By leveraging the star activity characteristic of nickases (mismatch cleavage), they designed a primer probe set with a nickase-cleavage recognition sequence. This allows for a cyclic process in which a template chain continuously binds to the probe and is continuously cleaved, releasing a fluorescent signal. This produces a stronger fluorescent signal and improves detection sensitivity. Although this technical method can increase the fluorescent signal intensity and sensitivity of isothermal amplification detection, it requires the inclusion of specific mismatch sequences in the target sequence, so its scope of application is somewhat limited.

[0008] Therefore, how to improve the nicking enzyme-based isothermal nucleic acid amplification detection technology, reduce nonspecific amplification, improve detection sensitivity and specificity, and achieve nucleic acid detection with fast speed, high sensitivity, good specificity, high accuracy and wider applicability is a difficult problem that needs to be overcome urgently.

[0009] Summary of the Invention

[0010] To solve the above problems, the present invention provides a novel nucleic acid isothermal amplification detection system and detection method thereof. By combining the use of a nicking endonuclease, a DNA polymerase (reverse transcriptase is added for RNA templates) and a ribonuclease, primers containing a nicking recognition sequence of the nicking endonuclease and a probe containing at least one ribonucleotide base are designed, and base modification is performed on the primers and probes. Thus, during the isothermal amplification process, the signal intensity of the target nucleic acid is continuously amplified, significantly improving the detection sensitivity, specificity and accuracy, while further shortening the detection time, improving the detection efficiency, preventing missed detection and false detection, and broadening the scope of application.

[0011] On the one hand, the present invention provides a nucleic acid isothermal amplification detection system, which includes primers, probes, nicking endonucleases and ribonuclease endonucleases; the primers contain a nicking recognition sequence for the nicking endonuclease; the probes contain at least one ribonucleotide base, and the ribonucleotide base can be complementary to the target sequence.

[0012] Furthermore, the system also includes DNA polymerase or reverse transcriptase; when the target sequence is a DNA sequence, it also includes DNA polymerase; when the target sequence is an RNA sequence, it also includes reverse transcriptase.

[0013] The present invention provides a novel isothermal amplification detection system for nucleic acids (including DNA and RNA) based on a nicking endonuclease, DNA polymerase (with the addition of reverse transcriptase for RNA templates), and endoribonuclease. Rapid nucleic acid amplification detection is achieved through the design of primer probes and repeated cyclic amplification under the combined action of the nicking endonuclease, endoribonuclease, and nucleic acid polymerase. The design of the primer probes primarily includes the following two aspects:

[0014] 1. Using the base sequence of the complementary strand of the nicking enzyme recognition sequence, a primer set with a nicking enzyme recognition sequence is designed. During the isothermal amplification process, the nicking enzyme cuts at a specific position in the primer region of the amplified fragment. Under the action of DNA polymerase, the nicked fragment continues to extend along the template, and the other template chain continues to bind to the primer, extend, and be cleaved to release the amplicon in a cycle.

[0015] 2. By utilizing the property of endoribonuclease that can hydrolyze RNA in DNA / RNA hybrids, a probe containing ribonucleotide bases is designed. During the isothermal amplification process, the probe is cut by the endoribonuclease, realizing a cyclic process in which the template chain continuously binds to the probe and is continuously cut to release a fluorescent signal, effectively increasing the fluorescence intensity of the target nucleic acid to be tested, improving the detection sensitivity, and further shortening the detection time.

[0016] In the existing nicking enzyme constant temperature amplification, one template chain can only bind to one fluorescent probe, and the fluorescent signal is low, especially in the early stage of amplification, making it difficult to quickly obtain detection results. For example, when using a molecular beacon probe, according to the luminescence mechanism of the molecular beacon fluorescent probe, the molecular beacon fluorescent probe is a U-shaped hairpin structure before binding to the template chain, and the fluorescent groups and quenching groups at both ends are close to each other and cannot emit fluorescence. When the fluorescent probe binds to the amplified product, the U-shaped hairpin structure opens into a straight chain, and the fluorescent group and the quenching group are separated, thereby emitting fluorescence. As the amplification continues, the fluorescent probe will fall off during the process of the amplified product changing from a single chain to a double chain, so that the fluorescent group and the quenching group are close to each other again, and the fluorescence disappears again. Therefore, the sensitivity of nucleic acid detection in the existing nicking enzyme constant temperature amplification is low, and it is difficult to achieve accurate detection.

[0017] The probes provided by the present invention (such as fluorescent probes) are used for constant temperature amplification. Since the fluorescent probes are provided with endoribonuclease recognition sites, the amplified products do not contain ribonucleotides and are not cleaved by endoribonucleases. Therefore, once the fluorescent probes bind to the single strand of the amplified product to form a double strand, they are recognized and cleaved by the endoribonuclease. Only after cleavage and detachment does the probe emit fluorescence (the fluorescent group of the probe is completely separated from the quenching group). New fluorescent probes continue to bind to the single strand of the amplified product. This cycle repeats, with the probes being continuously cleaved and releasing fluorescent signals, thereby continuously amplifying the fluorescent signal of the template strand and significantly improving the detection sensitivity.

[0018] Therefore, compared with the existing method of releasing a fluorescent signal by combining a probe with a template chain, the method provided by the present invention can effectively improve the fluorescence intensity and detection sensitivity of the target nucleic acid to be detected, and can also broaden the scope of application.

[0019] Existing nicking enzyme constant temperature amplification also has the problem of non-specific amplification, which can lead to missed detection and false detection. Because DNA polymerase has the possibility of DNA synthesis independent of primers or templates, in the nicking enzyme constant temperature amplification system, DNA polymerase can synthesize short nicking enzyme or endoribonuclease recognition sequences or similar repeated tandem fragments. These fragments are then rapidly amplified and extended to form a large number of non-specific amplification products, which can lead to missed detection and false detection.

[0020] The present invention also helps to prevent non-specific amplification and improve the specificity of the detection results by adopting a combination of nicking enzyme, DNA polymerase, and endoribonuclease. The reason may be that the endoribonuclease itself also has a high recognition specificity. Combined with the corresponding probe containing ribonucleotide bases, it can further specifically identify whether the amplified product is the target sequence.

[0021] In some embodiments, the nucleic acid isothermal amplification detection system provided herein includes the primer-probe set, which includes an upstream primer, a downstream primer, and a probe, wherein both the upstream primer and the downstream primer contain a nicking enzyme cleavage recognition sequence. Because nicking enzyme isothermal amplification involves nicking the primers to extend the template strand, both the upstream primer and the downstream primer must contain a nicking enzyme recognition sequence.

[0022] When the target sequence is a DNA sequence, the nucleic acid isothermal amplification detection system further includes a DNA polymerase; the temperature of the isothermal amplification is 50 to 60°C. The DNA polymerase (Bst DNA polymerase), nicking endonuclease Nt.BstNBI and ribonuclease RNase H2 provided by the present invention can function better at a temperature of 50 to 60°C, thereby performing highly efficient nicking enzyme isothermal amplification and enabling highly sensitive nucleic acid detection. Therefore, in the system provided by the present invention, the combination of DNA polymerase, nicking enzyme Nt.BstNBI and RNase H2 enzyme is just right, and the amplification time only takes 5 to 8 minutes.

[0023] Furthermore, the nicking endonuclease includes any one or more of Nt.BspQI, Nb.BbvCi, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Bpul 0l, Nt.Bpul 0l or N.SBspD61.

[0024] Each nickase has a specific recognition sequence. Once a single strand containing this recognition sequence binds to a complementary strand to form a double strand, the enzyme can cleave the strand containing the recognition sequence (in other words, a double strand must be formed before the strand containing the recognition sequence can be cleaved). The cleavage site is near the recognition sequence. The recognition sequence and cleavage position may vary between different nickases. For example, Nt.BstNBI cleaves a single strand containing the recognition sequence four nucleotides from the 3' end of the recognition sequence.

[0025] Theoretically, any nicking enzyme can be used in the isothermal nucleic acid amplification detection system of the present invention. Because different nicking enzymes have different cleavage recognition sequences, the present invention uniformly represents the cleavage recognition sequence of any nicking enzyme as "5'-NNNNNNNNNN-3'," where N is any of the four nucleotide bases A, C, T, and G. The number of Ns varies depending on the recognition sequence of the different nicking enzymes. However, the sensitivity, specificity, and accuracy of the detection systems constructed using different nicking enzymes may vary.

[0026] In some embodiments, the nicking enzyme is Nt.BstNBI, and its nicking recognition sequence is "5'-GAGTC-3'".

[0027] In some embodiments, the primers (including upstream primers and downstream primers) must contain "5'-GAGTC-3'".

[0028] In some embodiments, the recognition and cleavage sites of the nickase Nt.BstNBI are as follows:

[0029] 5'-N...NNNGAGTCNNNN...N-3'

[0030] 3'-N...NNNCTCAGNNNN...N-5'

[0031] Wherein, N=A, C, G or T, and the cleavage site sequence is 5'-GAGTC-3'.

[0032] Furthermore, the endoribonuclease is RNase H2.

[0033] Three main types of RNaseH have been discovered in nature: RNaseH1, RNaseH2, and H3. RNase H1 is typically used to degrade RNA templates after synthesizing complementary DNA (cDNA) via reverse transcription. It can also be used to cleave specific RNA sequences in the presence of short complementary DNA fragments. RNase H2 can be used to degrade RNA primer components of Okazaki fragments or introduce single-stranded cleavage at positions containing ribonucleotides. PCR experiments using thermostable RNase H2 have been reported. RNase H3 is more structurally similar to H2 but its activity is closer to H1.

[0034] There are three forms of RNA / DNA complexes: 1) RNA containing a single ribonucleotide within double-stranded DNA; 2) RNA consisting of one RNA strand and one DNA strand; and 3) RNA / DNA hybrid fragments consisting of four or more consecutive ribonucleotides.

[0035] RNase H2 can specifically recognize all of the above RNA / DNA complexes and exercise endonuclease activity; RNase H1 and H3 can recognize the second and third RNA / DNA complexes above and exercise cutting activity, but there may be off-target risks; some RNase H3 can also cut the first complex, but its efficiency is low. RNase H2 nuclease can recognize single ribonucleotide incorporation in the DNA double helix and cut at the 5' end of the ribonucleotide, so RNase H2 is preferably used as the endoribonuclease in the nucleic acid isothermal amplification detection system of the present invention. It is understandable that endoribonucleases with similar activities to RNase H2 can be used to construct the nucleic acid isothermal amplification detection system provided by the present invention.

[0036] The present invention, based on the recognition and cleavage activity of RNase H2, uses the reverse complement of the target sequence as the probe recognition sequence and selects 1-30 deoxyribonucleotide bases in this sequence to replace them with corresponding ribonucleotide bases. During the recognition process, the fluorescent probe will form a complementary pair with the specifically amplified product in the amplicon. At the same time, because the probe contains ribonucleotide bases, the probe bound to the target will be recognized and cleaved by RNase H2. After cleavage, a section of the probe "falls off" from the template, at which point other probes can bind to the template again. Therefore, a single template strand may bind to multiple probes, and the probes will be cleaved by RNase H2 to release fluorescent signals. This cycle is repeated, allowing each template strand to produce a stronger fluorescent response.

[0037] The probe and isothermal amplification method provided by the present invention are suitable for RNase H2 from prokaryotic and eukaryotic organisms. Since the cleavage sites of different RNase H2s are also different, the present invention uniformly represents the cleavage recognition sequence of any RNase H2 as "5'-rN-3'", where rN is any one of the four ribonucleotide bases A, C, U, and G, and the number of Ns varies accordingly according to the recognition sequence of different RNase H2s.

[0038] In the amplification embodiment, the present invention introduces one or more RNase H2 cleavage recognition sites into the probe. These recognition sites can be any combination of one or more of the ribonucleotide bases U, A, C, and G, collectively represented herein as rN. The probe sequence is "5'-N...NNNNrNNNNN...N-3'," where rN can be anywhere in the sequence and can be one or more. During the recognition process, the fluorescent probe will form a complementary pair with the specific amplification product. Simultaneously, due to the presence of ribonucleotide bases on the probe, the probe bound to the target is recognized and cleaved by RNase H2, generating a fluorescent signal.

[0039] Furthermore, the primer comprises a stabilizing region sequence, a nicking enzyme recognition sequence, and a target nucleic acid recognition region from the 5' end to the 3' end (see Figure 1); the probe is any one of a Taqman probe, an MGB probe, and a molecular beacon probe; and the label on the probe is any one of fluorescein, biotin, digoxigenin, and an isotope.

[0040] Studies have shown that RNase H2 nuclease has excellent cleavage activity in the nucleic acid isothermal amplification detection system provided by the present invention. Probes containing ribonucleotide bases can bind to single-stranded template DNA to form double strands and be cleaved by the RNase H2 enzyme, generating a strong fluorescent signal. However, when there is no template matching the probe in the reaction system, the RNase H2 enzyme cannot cleave.

[0041] It is understandable that the present invention is applicable to any probe, and studies have shown that the probe design provided by the present invention can significantly improve the detection sensitivity of nucleic acids.

[0042] Furthermore, the probe type is a molecular beacon probe.

[0043] Furthermore, the label on the probe is fluorescein, and both ends of the probe are modified with a fluorescent group or a quenching group respectively.

[0044] When one end of the fluorescent probe is modified with a fluorescent group, the other end is modified with a quencher group; or when one end is modified with a quencher group, the other end is modified with a fluorescent group.

[0045] In some embodiments, the fluorescent group is one or more fluorescent groups such as FAM, TET, VIC, HEX, TAMRA, ROX, Cy5, LCRED640, etc.; the quenching group is one or more quenching groups such as Dabcyl, BHQ1, BHQ2, BHQ3, MGB, etc.

[0046] Furthermore, the primer and / or probe contains base modifications, and the base modifications include any one or more of 2'-O-methyl, thio modification, alkyl, amino, and deoxyhypoxanthine modification.

[0047] The isothermal amplification detection system provided by the present invention may also have the problem of non-specific amplification when a nicking endonuclease, an endoribonuclease and a nucleic acid polymerase are used in combination, thereby leading to missed detection and false detection. This is because DNA polymerase has the possibility of synthesizing DNA independently of primers or templates. When an endoribonuclease or a nicking enzyme is present at the same time, this non-template or primer-dependent synthesis is effectively promoted. That is, when a nicking enzyme or an endoribonuclease is present, the DNA polymerase can synthesize short nicking enzyme or endoribonuclease recognition sequences or similar repeated tandem fragments, which are then rapidly amplified and extended to form a large number of non-specific amplification products, thereby leading to missed detection and false detection.

[0048] The present invention also helps to avoid mismatches by introducing specific base modifications in primers and probes to reduce non-specific amplification and improve the accuracy and specificity of detection results.

[0049] Furthermore, the base modification is a 2'-O-methyl modification, and the base at the 3' end of the primer and / or the upstream and downstream bases of the probe ribonucleic acid base site contain base modifications.

[0050] Studies have shown that compared with other base modification methods, 2'-O-methyl modification is more effective in improving the accuracy and specificity of detection results.

[0051] Since the amplification and extension of the primer starts from the 3' end, the base at the 3' end of the primer needs to be modified.

[0052] Since the probe used is a molecular beacon probe containing ribonucleic acid bases, the probe recognizes the complementary chain of the target sequence of the amplified product and combines with it to form a DNA / RNA hybrid chain. At the same time, when the ribonuclease cuts the hybrid chain, the ribonucleic acid base probe emits fluorescence. Therefore, the bases upstream and downstream of the ribonucleic acid base site of the probe need to be modified to avoid mismatches.

[0053] The specific number of bases that require base modification in primers and / or probes needs to be determined through specific experimental procedures. Because the number of bases that require base modification may vary for primers or probes with different sequences, it is necessary to determine the number through experimental screening.

[0054] Furthermore, some bases at specific positions of the primers and probes contain base modifications.

[0055] Studies have shown that 2'-O-methyl modification of both primers and probes does not affect the cleavage activity of nickases and endoribonucleases. Furthermore, compared to 2'-O-methyl modification of only primers or only probes, 2'-O-methyl modification of both primers and probes can more effectively prevent mismatches and nonspecific amplification, thereby improving amplification efficiency.

[0056] In some embodiments, the 1st to 11th bases at the 3' end of the upstream primer contain a 2'-O-methyl modification, and the 1st to 11th bases at the 3' end of the downstream primer contain a 2'-O-methyl modification; the -1st to -6th bases upstream and the +1st to +6th bases downstream of the probe ribonucleic acid base contain a 2'-O-methyl modification.

[0057] In some embodiments, the present invention provides a nucleic acid isothermal amplification detection system for Mycoplasma pneumoniae, comprising a primer probe set and a nicking endonuclease, a DNA polymerase, and an endoribonuclease, wherein the primer probe set comprises:

[0058] (1) MMP-16S-F (upstream primer): 5′-CGAACGGGTGAGTCACACGTATCCAATCTG-3′ (SEQ ID NO. 1);

[0059] (2) MMP-16S-R (downstream primer): 5′-CTCCCGTAGGAGTCTGGGCCGTGTCTCAG-3′ (SEQ ID NO. 2);

[0060] (3) Probe MMP-BPr: 5′-ROX-GTCATCACTA-rG-CTAATACCGATGAC-BHQ2-3′ (SEQ ID NO. 3).

[0061] The probe type is a molecular beacon probe, and both ends of the probe are modified with fluorescent groups or quenching groups; the endoribonuclease is RNase H2 enzyme.

[0062] The corresponding template target nucleic acid sequence recognized by the probe MMP-BPr is 5'-NNNNNGGTATTAGCTAGTNNNNN-3'. The probe can complementarily pair with the template. The RNase H2 enzyme recognizes the rG ribonucleotide base on the probe and cuts it, while the template target sequence will not be cut.

[0063] This system can be used for high-sensitivity detection of the 16S sequence of Mycoplasma pneumoniae.

[0064] Furthermore, the primer probe of the 16S gene detection system of Mycoplasma pneumoniae was modified with 2'-O-methyl, which did not affect the function of the probe and the detection of Mycoplasma pneumoniae. The sequences of the primer and probe after 2'-O-methyl modification are as follows:

[0065] MMP-16S-mF: 5'-CGAACGGGTGAGTCACACGmTmAmTmCmCmAmAmTCTG-3' (SEQ ID NO. 4);

[0066] MMP-16S-mR: 5'-CTCCCGTAGGAGTCTGGGCmCmGmTmGmTmCmTmCAG-3' (SEQ ID NO.5);

[0067] MMP-16S-mBPr: 5′-ROX-GTCATCmAmCmTA-rG-CmTmAmATACCGATGAC-BHQ2-3′ (SEQ ID NO. 6) (wherein the prefix m indicates that the base has been modified with 2′-O-methyl).

[0068] This system can be used for high-sensitivity, high-accuracy and high-specificity detection of the 16S sequence of Mycoplasma pneumoniae.

[0069] In some embodiments, the present invention provides a nucleic acid isothermal amplification detection system for a novel coronavirus, comprising a primer probe set and a nicking endonuclease, a DNA polymerase, and an endoribonuclease, wherein the primer probe set comprises:

[0070] (1) COV-SF (upstream primer): 5′-ACCATACAGAGTCGAGTAGTACTTTCTTTTG-3′ (SEQ ID NO. 7);

[0071] (2) COV-SR (downstream primer): 5′-CCAAGTAGGAGTCAGTTGATCTGCATGA-3′ (SEQ ID NO. 8);

[0072] (3) Probe COV-S-BPr: 5'-FAM-GGTGCAT-rA-TGTCAATTTCAACTGCACC-BHQ1-3' (SEQ ID NO. 9).

[0073] This system can be used for high-sensitivity detection of the S gene of the new coronavirus.

[0074] This system can be used for high-sensitivity, high-accuracy and high-specificity detection of the S gene of the new coronavirus.

[0075] On the other hand, the present invention provides a nucleic acid isothermal amplification detection method, which uses the nucleic acid isothermal amplification detection system as described above to perform nucleic acid isothermal amplification detection.

[0076] On the other hand, the present invention provides the use of 2'-O-methyl modification for preparing a reagent for improving the accuracy and specificity of a nucleic acid isothermal amplification detection system for target sequence detection, wherein the nucleic acid isothermal amplification detection system includes a primer, a probe, a nicking endonuclease, a DNA polymerase and a ribonuclease; the primer contains a nicking recognition sequence for the nicking endonuclease; the probe contains at least one ribonucleotide base, and the ribonucleotide base is complementary to the target sequence.

[0077] On the other hand, the present invention provides the use of a nicking enzyme in preparing a reagent for improving the detection sensitivity of a nucleic acid isothermal amplification detection system, wherein the nucleic acid isothermal amplification detection system includes a primer, a probe, a nicking endonuclease, a DNA polymerase and a ribonuclease; the primer contains a nicking recognition sequence of the nicking endonuclease; the probe contains at least one ribonucleotide base, and the ribonucleotide base can be complementary to the target sequence.

[0078] On the other hand, the present invention provides a use of a probe as a reagent for improving the detection sensitivity of a nucleic acid isothermal amplification detection system, wherein the nucleic acid isothermal amplification detection system includes a primer, a probe, a nicking endonuclease, a DNA polymerase and a ribonuclease; the primer contains a nicking recognition sequence of the nicking endonuclease; the probe contains at least one ribonucleotide base, and the ribonucleotide base can be complementary to the target sequence.

[0079] On the other hand, the present invention provides a use of a nicking enzyme and a ribonuclease for preparing a highly sensitive nucleic acid isothermal amplification detection system, wherein the nucleic acid isothermal amplification detection system comprises a primer, a probe, a nicking endonuclease, a DNA polymerase and an endonuclease; the primer contains a nicking recognition sequence of the nicking endonuclease; the probe contains at least one ribonucleotide base, and the ribonucleotide base can be complementary to the target sequence.

[0080] The nucleic acid isothermal amplification detection system provided by the present invention has the following beneficial effects:

[0081] 1. By combining the activity characteristics of Bst DNA polymerase, Nt.BstNBI nickase, and RNase H2 endoribonuclease, we designed and developed an isothermal detection reaction system for rapid nucleic acid detection based on enzyme amplification and fluorescent probe secondary signal cascade amplification. Furthermore, the addition of reverse transcriptase to the system allows for the amplification and detection of RNA nucleic acids.

[0082] 2. By introducing endoribonuclease into the nickase amplification detection system, the detection efficiency, sensitivity and specificity are significantly improved, and the detection time is only 5 to 8 minutes;

[0083] 3. The cleavage activity of RNase H2 nuclease was verified. The probe containing ribonucleotide bases can bind to the single-stranded template DNA to form a double strand and be cleaved by RNase H2 enzyme, generating a strong fluorescent signal. When there is no template matching the probe in the reaction system, RNase H2 enzyme cannot cleave.

[0084] 4. By modifying the bases of primers and probes, the non-specific amplification problem of the nucleic acid isothermal amplification detection system is solved, significantly improving the accuracy and specificity of the test results and avoiding missed detection and false detection;

[0085] 5. The application of this nucleic acid isothermal amplification detection system in POCT products has huge market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 is a schematic diagram of the primer design principle;

[0087] Figure 2 is a schematic diagram of the probe design principle;

[0088] FIG3 is a schematic diagram of the detection results of different probe sequences in Example 1;

[0089] Figure 4 is a schematic diagram of the sensitivity results of novel coronavirus nucleic acid detection in Example 2;

[0090] FIG5 is a schematic diagram of the test results of different primer probes used in Example 3 for Mycoplasma pneumoniae samples with different concentrations;

[0091] FIG6 is a schematic diagram showing the specific results of the circulating probe cleavage amplification nucleic acid detection of Mycoplasma pneumoniae in Example 3. DETAILED DESCRIPTION

[0092] The preferred embodiments of the present invention are described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate understanding of the present invention and do not limit it in any way. The raw materials and equipment used in the specific embodiments of the present invention are all known products and were obtained by purchasing commercially available products.

[0093] In the following examples, the nicking enzyme used was Nt.BstNBI nicking enzyme purchased from NEB, product catalog number #R0607S, the DNA polymerase used was Bst DNA polymerase purchased from Thermo Fisher Scientific Inc., product catalog number A56656, RNase H2 enzyme was purchased from Integrated DNA Technologies, Inc., product catalog number #11-03-02-03, and RTx reverse transcriptase was purchased from NEB, product catalog number #M0380L.

[0094] Example 1: Verification of the recognition and cleavage activity of RNase H2 enzyme

[0095] This example uses RNase H2 as an example to experimentally verify the cleavage characteristics of RNase H2. Specifically, when a probe containing ribonucleotide bases forms a DNA / RNA hybrid with a target, the RNase H2 enzyme will cleave it. However, if the probe containing ribonucleotide bases does not recognize a complementary nucleic acid, the RNase H2 enzyme will not cleave it.

[0096] Based on the RNase H2 enzyme cleavage recognition sequence (5'-NNNNN-rN-NNNN-3', where N represents any deoxyribonucleotide base and rN represents any ribonucleotide base) and the molecular beacon probe design rules, two probes containing different numbers of ribonucleotides were designed. The design principle is shown in Figure 2. The fluorophore FAM and the quencher BHQ1 were labeled at the 5' and 3' ends of the probe sequence, respectively, as follows:

[0097] ASV-NN-R3Probe1: 5'-FAM-TGTGTTACTTTGAGTrCAAATCGAAGAAACACA-BHQ1-3' (SEQ ID NO. 10);

[0098] ASV-NN-R3Probe2: 5'-FAM-TGTGTTACTTTGAGrUrCrArArArUrCrGAAGAAACACA-BHQ1-3' (SEQ ID NO. 11);

[0099] The target recognition sequence corresponding to this probe is:

[0100] ASV-TR7: 5'-TGCGTTTCTTCGATTT-GACTC-AAAGTAAACAG-3' (single-stranded template) (SEQ ID NO. 12).

[0101] The above-mentioned probe and single-stranded template DNA fragment were synthesized, and cleavage reaction was carried out using RNase H2 enzyme. The specific reaction system is shown in Table 1.

[0102] Table 1. RNase H2 enzyme digestion reaction system and components

[0103] The experimental design is as follows: 1: ASV-NN-R3Probe1 probe and ASV-TR7 are used as templates in the reaction system; 2: ASV-NN-R3Probe2 probe and ASV-TR7 are used as templates in the reaction system; 3: ASV-NN-R3Probe1 probe and water are used as templates in the reaction system; 4: ASV-NN-R3Probe2 probe and water are used as templates in the reaction system.

[0104] A fluorescence thermostatic amplification instrument (GS8) was used for reaction and real-time detection. The reaction conditions were 60°C, the duration was 30 minutes, and the fluorescence acquisition period was 30 seconds per point.

[0105] The experimental results are shown in Figure 3. It can be seen from the results in the figure that the probe sequence can bind to the ASV-TR7 single-stranded template DNA to form a double strand and be cut by the RNase H2 enzyme, generating a strong fluorescent signal (1 and 2 in Figure 3); however, when there is no template matching the probe in the reaction system, the RNase H2 enzyme cannot cut (3 and 4 in Figure 3).

[0106] It should be noted that, in addition to RNase H2, other nucleases have similar cleavage characteristics, and probes containing the enzyme cleavage sites of these nucleases can also achieve this function and fall within the scope of protection of this application.

[0107] Example 2: Detection of novel coronavirus

[0108] According to the nucleic acid isothermal amplification detection technology of the present invention, the primer probe design principle, the new coronavirus S gene sequence, and the Nt.BstNBI, Nt.BspD6I nickase and RNase H2 enzyme characteristics, the primers and probes for specific new coronavirus S gene detection are designed and synthesized, wherein the design rules of the probe are shown in Figure 2, including a recognition region that can bind to the target chain, an RNase H2 enzyme recognition site, and during the nickase isothermal amplification process, the primer rapidly amplifies the detection region sequence under the action of the nickase and the polymerase. After the probe recognizes the target sequence, it combines to form a double chain. The RNase H2 enzyme hydrolyzes the RNA base in the substrate in the hybrid chain. Under the joint action of the nicking endonuclease, the ribonuclease and the nucleic acid polymerase, rapid nucleic acid amplification detection is achieved. This embodiment is by screening a suitable target segment, and then designs multiple pairs of primer probes. The recognition region, nickase recognition site, and stable region in different primers are different. After multiple screenings, the optimal primer sequence (upstream primer and downstream primer) as shown below is finally obtained, and the influence of different probe sequences on the nucleic acid detection results is investigated, as follows:

[0109] COV-SF: 5'-ACCATACAGAGTCGAGTAGTACTTTCTTTTG-3'(SEQ ID NO.7)

[0110] COV-SR: 5'-CCAAGTAGGAGTCAGTTGATCTGCATGA-3' (SEQ ID NO.8)

[0111] COV-S-BPr: 5'-FAM-GGTGCAT-rA-TGTCAATTTCAACTGCACC-BHQ1-3'(SEQ ID NO.9)

[0112] The purchased novel coronavirus S gene RNA standard material (No.: NIM-RM5208, China National Institute of Metrology) was used as a template for testing and detection. The specific implementation steps are as follows:

[0113] Step 1: In 8 PCR reaction tubes, prepare the isothermal amplification detection system according to Table 2.

[0114] Table 2. Novel Coronavirus Nucleic Acid Isothermal Amplification Detection System

[0115] Step 2: Use TE Buffer to dilute the 200-novel coronavirus S gene RNA standard material to 200 copies / μl, 40 copies / μl, 20 copies / μl, 10 copies / μl, 5 copies / μl, 2 copies / μl, and 1 copy / μl.

[0116] Step 3: Add 5 μl of different concentrations of SARS-CoV-2 S gene RNA standard material to the 8-tube PCR reaction strip prepared in step 1 to dilute the template;

[0117] Step 4: After shaking and mixing the 8-tube PCR reaction containing the complete reagent system, place it in a fluorescence thermostatic amplification instrument (GS8) for reaction and real-time detection. The reaction conditions are 50°C, the duration is 10 minutes, and the fluorescence acquisition cycle is 15 seconds / point.

[0118] 2. Experimental results

[0119] The experimental results are shown in Figure 4. The copy numbers of samples 1 to 8 are 1000 copies, 200 copies, 100 copies, 50 copies, 25 copies, 10 copies, 5 copies, and negative, respectively. As shown in Figure 4, when 5-1000 copies of the novel coronavirus S gene RNA standard material are used, the isothermal amplification reaction has a higher fluorescence response, indicating that the method in this embodiment can detect the novel coronavirus S gene RNA standard material. The novel coronavirus S gene RNA standard material is RNA. Therefore, this application is based on the nucleic acid isothermal amplification detection technology to perform amplification detection of the novel coronavirus S gene RNA, and the detection sensitivity of the novel coronavirus S gene RNA reaches 1000 copies / ml (converted according to 1 copy / ul of the RNA template loading amount 5ul, i.e. 1000 copies / 1 ml).

[0120] Example 3: Application of the nucleic acid isothermal amplification detection system in the detection of Mycoplasma pneumoniae nucleic acid

[0121] This example examines the following four types of detection systems for Mycoplasma pneumoniae:

[0122] 1. In the existing nicking enzyme amplification detection system (nicking enzyme and DNA polymerase), the primers contain the nicking recognition sequence of the nicking endonuclease but no RNase H2, and the probes do not have an RNase H2 cleavage site.

[0123] 2. The existing PCR detection system (DNA polymerase, no nicking enzyme) contains a nicking endonuclease cleavage recognition sequence in the primer, but no RNase H2, and the probe has no RNase H2 cleavage site;

[0124] 3. Nicking enzyme, DNA polymerase and RNase H2. The primer contains the nicking recognition sequence of the nicking endonuclease, and the probe contains the RNase H2 cleavage site.

[0125] 4. Nicking enzyme, DNA polymerase and RNase H2. The primer contains the nicking recognition sequence of the nicking endonuclease, and the probe contains the RNase H2 cleavage site. Both the primer and the probe are 2'-O-methyl-modified.

[0126] The experimental method is as follows:

[0127] In this example, suitable target segments were screened and multiple pairs of primers and probes were designed. Different primers had different recognition regions, nickase recognition sites, and stabilization regions. After multiple rounds of screening, the optimal primer sequences (upstream and downstream primers) were finally obtained, as shown below. Based on the primer and probe design principles of cyclic amplification detection reaction technology and the Mycoplasma pneumoniae nucleic acid sequence, four different types of Mycoplasma pneumoniae nucleic acid detection primers and probes were designed and synthesized, and then nucleic acid isothermal amplification detection was performed. The primers and probes for the four detection systems are as follows:

[0128] The first one:

[0129] MMP-16S-F: 5'-CGAACGGGTGAGTCACACGTATCCAATCTG-3' (SEQ ID NO. 1);

[0130] MMP-16S-R: 5'-CTCCCGTAGGAGTCTGGGCCGTGTCTCAG-3' (SEQ ID NO. 2);

[0131] MMP-16S-BP0: 5'-ROX-GTCATCACTAGCTAATACCGATGAC-BHQ2-3' (SEQ ID NO. 13);

[0132] The second type:

[0133] MMP-16S-F0: 5'-CGAACGGGTGAGTAACACGTATCCAATCTG-3' (SEQ ID NO. 22);

[0134] MMP-16S-R0: 5'-CTCCCGTAGGAGTATGGGCCGTGTCTCAG-3' (SEQ ID NO. 23);

[0135] MMP-16S-BPr:5'-ROX-GTCATCACTA-rG-CTAATACCGATGAC-BHQ2-3' (SEQ ID NO.3);

[0136] The third type:

[0137] MMP-16S-F: 5'-CGAACGGGTGAGTCACACGTATCCAATCTG-3' (SEQ ID NO. 1);

[0138] MMP-16S-R: 5'-CTCCCGTAGGAGTCTGGGCCGTGTCTCAG-3' (SEQ ID NO. 2);

[0139] MMP-16S-BPr:5'-ROX-GTCATCACTA-rG-CTAATACCGATGAC-BHQ2-3' (SEQ ID NO.3);

[0140] The fourth type:

[0141] MMP-16S-mF: 5'-CGAACGGGTGAGTCACACGmTmAmTmCmCmAmAmTCTG-3' (SEQ ID NO. 4);

[0142] MMP-16S-mR: 5'-CTCCCGTAGGAGTCTGGGCmCmGmTmGmTmCmTmCAG-3' (SEQ ID NO.5);

[0143] MMP-16S-mBPr: 5'-ROX-GTCATCmAmCmTA-rG-CmTmAmATACCGATGAC-BHQ2-3' (SEQ ID NO. 6);

[0144] Inactivated Mycoplasma pneumoniae culture (1×10 9 CCU / ml, No. 20210603, manufactured by the Veterinary Research Institute of Jiangsu Academy of Agricultural Sciences) was used as a template for cyclic amplification detection reaction. The specific experimental steps are as follows:

[0145] Step 1: In a PCR reaction tube, prepare the cyclic amplification detection system according to Table 2;

[0146] Step 2: Use TE Buffer to serially dilute the inactivated Mycoplasma pneumoniae culture to 5000 CCU / mL, 500 CCU / mL, and 50 CCU / mL;

[0147] Step 3: 1 ml of inactivated Mycoplasma pneumoniae culture at different concentrations was taken for nucleic acid extraction using a DNA / RNA nucleic acid extraction kit (TianGen, DF438-01) and dissolved in 30 μL of TE solution;

[0148] Step 4: Add 5 μL of nucleic acid extracted in step 3 to the PCR reaction tube prepared in step 1;

[0149] Step 5: After vortexing and mixing the PCR reaction tube containing the complete reagent system, place it in a fluorescent quantitative PCR instrument (Thermofisher, QuantStudio 3) for reaction and real-time detection. The reaction conditions are: 55°C, duration of 20 minutes, fluorescence acquisition period of 30 seconds / point, and direct isothermal nucleic acid amplification detection reaction.

[0150] Table 3. Reagent system for detecting Mycoplasma pneumoniae by amplification detection method

[0151] The detection sensitivity of the four detection systems was investigated, and the experimental results are shown in FIG5 .

[0152] The results in Figure 5 show that the fluorescence response value of the nickase polymerase isothermal amplification reaction significantly increased when the MMP-16S-BPr probe was used compared to the MMP-16S-BP0 probe, indicating that the sensitivity of the nickase polymerase isothermal amplification reaction was significantly improved when the MMP-16S-BPr probe was used (the earlier the peak onset time, the higher the fluorescence response value, and the higher the sensitivity). The nucleic acid isothermal amplification detection reaction using the 2'-O-methyl-modified primer probe still had a high fluorescence intensity, enabling highly sensitive detection of Mycoplasma pneumoniae.

[0153] By comparing the four types of primer probes, it was found that compared with the first and second types, the MMP-16S-BPr (third detection system) or MMP-16S-mBPr (fourth detection system) probes contained the sequence "5'-NNNrNNNN-3'" containing ribonucleotide bases, which were complementary to the target sequence to form a double strand that would be recognized and cleaved by the RNase H2 enzyme. This indicates that the use of probes containing RNase H2 enzyme recognition sites can improve the sensitivity of the cyclic amplification detection reaction.

[0154] At the same time, by comparing the four types of primer probes, it was found that under the premise of the same sample to be tested, the positive peak time of MMP-16S-BPr or MMP-16S-mBPr probe was much earlier than that of MMP-16S-BP0, indicating that the use of probes containing RNase H2 enzyme recognition sites can significantly improve the detection speed of the cyclic amplification detection reaction.

[0155] As shown in Figure 5, using the fluorescent probes MMP-16S-BPr or MMP-16S-mBPr, which contain RNase H2 cleavage recognition sites, significantly increases the fluorescence value of nucleic acid detection results, thereby improving detection sensitivity. This means that both Detection Systems 3 and 4 can detect M. pneumoniae samples at concentrations of 50 CCU / mL. Detection Systems 1 and 2 can detect M. pneumoniae samples at concentrations of at least 500 CCU / mL and 5000 CCU / mL, respectively. See Table 4 for details.

[0156] Table 4 Sensitivity of different detection systems and corresponding average peak time schedules

[0157] Through the above analysis, it can be concluded that: compared with the first detection system, the detection sensitivity, positive peak onset speed and detection fluorescence intensity of the third and fourth detection systems with the addition of RNase H2 enzyme and a probe combination containing the corresponding recognition site were significantly improved, indicating that the use of RNase H2 enzyme and MMP-16S-BPr probe can significantly improve the detection sensitivity, detection efficiency and signal-to-noise ratio of the nucleic acid detection system; compared with the second detection system, the detection sensitivity, positive peak onset speed and detection fluorescence intensity of the third and fourth detection systems with the addition of Nt.BstNBI enzyme and a primer combination containing the corresponding recognition site were significantly improved, indicating that the use of a nicking enzyme and a primer combination containing the corresponding recognition site can significantly improve the detection sensitivity, detection efficiency and signal-to-noise ratio of the nucleic acid detection system; compared with the third detection system, the methylated primer-probe combination of the fourth detection system can also obtain comparable detection sensitivity and positive peak onset speed. In addition, the fluorescence intensity is also improved to a certain extent, indicating that the methylated primer-probe combination will not affect the detection effect of the third detection system.

[0158] The negative coincidence rates of the four detection systems were investigated. Ten throat swab samples from healthy individuals were extracted with a DNA / RNA nucleic acid extraction kit (TianGen, DF438-01) and dissolved in 30 μL of TE solution. These samples were used as negative samples to test the negative coincidence rates of the four detection systems. The test results are shown in Table 5.

[0159] Table 5. Negative test results of different detection systems

[0160] As can be seen from Table 5, the negative coincidence rates when using the first detection system, the second detection system, the third detection system and the fourth detection system are 80%, 90%, 100% and 100% respectively. Therefore, in terms of negative coincidence (i.e., false positive phenomenon), the first and second detection systems are not as good as the third and fourth detection systems, and false detection may occur. The reason may be that the RNase H2 enzyme can further improve the specificity of recognition.

[0161] The detection specificity of the fourth form of the detection system was investigated, using the nucleic acids of Mycoplasma pneumoniae, Klebsiella pneumoniae, Streptococcus pneumoniae, Ureaplasma urealyticum, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Mycoplasma hominis, Candida glabrata, Cryptococcus neoformans, Aspergillus fumigatus, Streptococcus pyogenes, Bordetella pertussis, Candida albicans, Mycobacterium tuberculosis, Legionella pneumophila, H5N1, and H3N2 as templates. The three detection systems described above were used for amplification, respectively. Among them, inactivated cross-experimental microorganisms such as Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Candida glabrata, Cryptococcus neoformans, Aspergillus fumigatus, Streptococcus pyogenes, Bordetella pertussis, Candida albicans, Mycobacterium tuberculosis, and Legionella pneumophila were purchased from Beijing Beina Chuanglian Biotechnology Research Institute, inactivated H5N1 and H3N2 strains were purchased from Shenzhen Haisian Biotechnology Co., Ltd., and inactivated strains of Ureaplasma urealyticum and Mycoplasma hominis were purchased from Guangzhou Bondsheng Biotechnology Co., Ltd.

[0162] The test results of the fourth form of the detection system are shown in Figure 6. Sample 19 is Mycoplasma pneumoniae nucleic acid, and samples 1 to 18 are Klebsiella pneumoniae, Streptococcus pneumoniae, Ureaplasma urealyticum, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Mycoplasma hominis, Candida glabrata, Cryptococcus neoformans, Aspergillus fumigatus, Streptococcus pyogenes, Bordetella pertussis, Candida albicans, Mycobacterium tuberculosis, Legionella pneumophila, H5N1, and H3N2, respectively. As shown in Figure 6, only the amplification detection reaction using Mycoplasma pneumoniae nucleic acid as a template can produce a fluorescent response. When other bacteria or viruses such as Klebsiella pneumoniae, Streptococcus pneumoniae, and Ureaplasma urealyticum are used as templates, there is no fluorescent response or the response is almost zero. This result shows that the nucleic acid amplification detection reaction of the present invention has good specificity and the test results are very accurate.

[0163] This embodiment also proves through research that for some complex viruses, the fourth form of detection system helps to better prevent nonspecific amplification and improve the specificity of the detection results, thereby preventing missed detections and false positives. For example, in the detection of African swine fever virus, nonspecific amplification will still occur if the primer probe set is not 2'-O-methyl-modified. However, after the primer probe set is 2'-O-methyl-modified, the nonspecific amplification problem will be completely solved, thereby improving the specificity of the detection results.

[0164] Example 4: Effects of Different Nicking Enzyme Types on Mycoplasma Pneumoniae Nucleic Acid Detection

[0165] This example uses the method provided in Example 3, employing the fourth type of primer-probe set for nucleic acid amplification. The nicking enzymes are replaced with the following: Nb.BsmI (recognition sequence 5'-GAATCG-3'), Nb.BsrDI (recognition sequence 5'-GCAATG-3'), Nt.BspQI (recognition sequence 5'-GCTCTTC-3'), and Nt.AlwI (recognition sequence 5'-GGTAC-3'). The corresponding nicking enzyme recognition sequences are replaced in the upstream and downstream primers, respectively. The effects of using different nicking enzymes on the isothermal nucleic acid amplification detection system provided by the present invention were investigated.

[0166] The primer and probe sequence information is as follows:

[0167] Nt.BstNBI:

[0168] MMP-16S-mF: 5'-CGAACGGGTGAGTCACACGmTmAmTmCmCmAmAmTCTG-3' (SEQ ID NO. 4);

[0169] MMP-16S-mR: 5'-CTCCCGTAGGAGTCTGGGCmCmGmTmGmTmCmTmCAG-3' (SEQ ID NO.5);

[0170] MMP-16S-mBPr: 5'-ROX-GTCATCmAmCmTA-rG-CmTmAmATACCGATGAC-BHQ2-3' (SEQ ID NO. 6);

[0171] Nb.BsmI:

[0172] MMP-16S-mF2: 5'-CGAACGGGGAATCGACACGmTmAmTmCmCmAmAmTCTG-3' (SEQ ID NO. 14);

[0173] MMP-16S-mR2: 5'-CTCCCGTAGAATCGTGGGCmCmGmTmGmTmCmTmCAG-3' (SEQ ID NO. 15);

[0174] Nb.BsrDI:

[0175] MMP-16S-mF3: 5'-CGAACGGGGCAATGACACGmTmAmTmCmCmAmAmTCTG-3' (SEQ ID NO. 16);

[0176] MMP-16S-mR3: 5'-CTCCCGTAGCAATGTGGGCmCmGmTmGmTmCmTmCAG-3' (SEQ ID NO. 17);

[0177] Nt.BspQI:

[0178] MMP-16S-mF4: 5'-CGAACGGGCTCTTCACACGmTmAmTmCmCmAmAmTCTG-3' (SEQ ID NO. 18);

[0179] MMP-16S-mR4: 5'-CTCCCGTGCTCTTCTGGGCmCmGmTmGmTmCmTmCAG-3'SEQ ID NO.19);

[0180] Nt.AlwI:

[0181] MMP-16S-mF5: 5'-CGAACGGGTGGTACACACGmTmAmTmCmCmAmAmTCTG-3' (SEQ ID NO. 20);

[0182] MMP-16S-mR5: 5'-CTCCCGTAGGGTACTGGGCmCmGmTmGmTmCmTmCAG-3' (SEQ ID NO. 21);

[0183] The test results are shown in Table 6.

[0184] Table 6. Effects of different nicking enzyme types on test results

[0185] As can be seen from Table 6, different nicking enzymes are basically applicable to the isothermal amplification of nucleic acids provided by the present invention. However, different nicking enzymes have different peak onset times and detection speeds (the earlier the peak onset time, the faster the detection speed), and there may be differences in detection sensitivity. The most preferred nicking enzyme is Nt.BstNBI.

[0186] Studies have shown that the nicking enzyme Nt.BstNBI is particularly suitable for the nucleic acid isothermal amplification detection system provided by the present invention. This is mainly due to the fact that, under the reaction conditions of this amplification detection system, Nt.BstNBI has the best optimal reaction temperature and the best enzyme activity under these detection reaction conditions. Experimental results show that Nb.BsmI, Nb.BsrDI, Nt.BspQI, and Nt.AlwI can all be used in this detection system, but there are certain differences in the detection speed and sensitivity. According to existing public data, this is mainly due to the different optimal reaction temperatures and enzyme activity efficiencies of different nicking enzymes.

[0187] In addition, this embodiment also designs corresponding primers and probes for the African swine fever B646L gene for use in nucleic acid isothermal amplification detection reactions, which also have very good detection sensitivity and specificity, with a detection sensitivity of 500 copies / ml.

[0188] The above results indicate that the method provided by the present invention for improving the primer specificity in a recombinase activity reaction can significantly improve the detection efficiency and detection specificity of the detection system and obtain higher detection sensitivity.

[0189] Although the above description has been given with general descriptions and preferred implementation cases, it is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A nucleic acid isothermal amplification detection system, characterized in that: The method comprises a primer, a probe, a nicking endonuclease and a ribonuclease; the primer contains a nicking endonuclease cutting recognition sequence; the probe contains at least one ribonucleotide base, and the ribonucleotide base can be complementary to the target sequence.

2. The nucleic acid isothermal amplification detection system according to claim 1, wherein: It also includes DNA polymerase or reverse transcriptase; when the target sequence is a DNA sequence, it includes DNA polymerase, and when the target sequence is an RNA sequence, it also includes reverse transcriptase in addition to DNA polymerase.

3. The nucleic acid isothermal amplification detection system according to claim 1, wherein: The primers include an upstream primer and a downstream primer, and both the upstream primer and the downstream primer contain a cutting recognition sequence of a nicking enzyme.

4. The nucleic acid isothermal amplification detection system according to claim 2, wherein: The nicking endonuclease includes any one or more of Nt.BspQI, Nb.BbvCi, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Bpul 0l, Nt.Bpul 0l or N.SBspD61.

5. The nucleic acid isothermal amplification detection system according to claim 4, wherein: The nicking endonuclease is Nt.BstNBI.

6. The nucleic acid isothermal amplification detection system according to claim 2, wherein: The endoribonuclease is RNase H2.

7. The nucleic acid isothermal amplification detection system according to claim 3, wherein: The primer comprises a stabilizing region sequence, a nicking enzyme recognition sequence, and a target nucleic acid recognition region from the 5' end to the 3' end; the probe is any one of a Taqman probe, an MGB probe, and a molecular beacon probe; and the label on the probe is any one of fluorescein, biotin, digoxigenin, and an isotope.

8. The nucleic acid isothermal amplification detection system according to claim 7, wherein: The probe type is a molecular beacon probe.

9. The nucleic acid isothermal amplification detection system according to claim 8, wherein: The label on the probe is fluorescein, and both ends of the probe are modified with a fluorescent group or a quenching group respectively.

10. The nucleic acid isothermal amplification detection system according to claim 9, wherein: The primer and / or probe contains base modifications, and the base modifications include any one or more of 2'-O-methyl, thio modification, alkyl, amino, and deoxyhypoxanthine modification.

11. The nucleic acid isothermal amplification detection system according to claim 10, wherein: The base modification is a 2'-O-methyl modification, and the base at the 3' end of the primer and / or probe contains a base modification.

12. The nucleic acid isothermal amplification detection system according to claim 11, wherein: The 1st to 11th bases at the 3' end of the upstream primer contain 2'-O-methyl modification, and the 1st to 11th bases at the 3' end of the downstream primer contain 2'-O-methyl modification; the -1st to -6th bases upstream and the +1st to +6th bases downstream of the probe ribonucleic acid base contain 2'-O-methyl modification.

13. A nucleic acid isothermal amplification detection system for Mycoplasma pneumoniae, characterized in that: The invention comprises a primer probe set, a nicking endonuclease, a DNA polymerase, and an endoribonuclease, wherein the primer probe set comprises: (1) an upstream primer having a nucleotide sequence as shown in SEQ ID NO. 1; (2) a downstream primer having a nucleotide sequence as shown in SEQ ID NO. 2; (3) A probe having a nucleotide sequence as shown in SEQ ID NO.

3.

14. The nucleic acid isothermal amplification detection system for Mycoplasma pneumoniae according to claim 13, wherein: The probe type is a molecular beacon probe, and both ends of the probe are modified with fluorescent groups or quenching groups; the endoribonuclease is RNase H2 enzyme.

15. The nucleic acid isothermal amplification detection system for Mycoplasma pneumoniae according to claim 14, characterized in that: The primer probe set has a 2'-O-methyl modification. The sequences of the primers and probes after 2'-O-methyl modification are as follows: Upstream primer: 5′-CGAACGGGTGAGTCACACGmTmAmTmCmCmAmAmTCTG-3′; Downstream primer: 5′-CTCCCGTAGGAGTCTGGGCmCmGmTmGmTmCmTmCAG-3′; Probe: 5′-ROX-GTCATCmAmCmTA-rG-CmTmAmATACCGATGAC-BHQ2-3′; The prefix "m" indicates that the base has been modified with 2'-O-methyl.

16. A novel coronavirus nucleic acid isothermal amplification detection system, characterized in that: The invention comprises a primer probe set, a nicking endonuclease, a DNA polymerase, and an endoribonuclease, wherein the primer probe set comprises: (1) an upstream primer having a nucleotide sequence as shown in SEQ ID NO. 7; (2) a downstream primer having a nucleotide sequence as shown in SEQ ID NO. 8; (3) A probe having a nucleotide sequence as shown in SEQ ID NO.

9.

17. A nucleic acid isothermal amplification detection method, characterized in that: Nucleic acid isothermal amplification detection is performed using the nucleic acid isothermal amplification detection system according to any one of claims 1 to 12, or the nucleic acid isothermal amplification detection system for Mycoplasma pneumoniae according to any one of claims 13 to 15, or the nucleic acid isothermal amplification detection system for the new coronavirus according to claim 16.

18. Use of 2'-O-methyl modification for preparing a reagent for improving the accuracy and specificity of target sequence detection in a nucleic acid isothermal amplification detection system, characterized in that: The nucleic acid isothermal amplification detection system comprises primers, probes, nicking endonucleases, DNA polymerases and ribonuclease endonucleases; the primers contain a nicking endonuclease cleavage recognition sequence; the probes contain at least one ribonucleotide base, and the ribonucleotide base can be complementary to the target sequence.

19. Use of a nicking enzyme in preparing a reagent for improving the detection sensitivity of a nucleic acid isothermal amplification detection system, characterized in that: The nucleic acid isothermal amplification detection system comprises primers, probes, nicking endonucleases, DNA polymerases and ribonuclease endonucleases; the primers contain a nicking endonuclease cleavage recognition sequence; the probes contain at least one ribonucleotide base, and the ribonucleotide base can be complementary to the target sequence.

20. Use of a probe in a reagent for improving the detection sensitivity of a nucleic acid isothermal amplification detection system, characterized in that: The nucleic acid isothermal amplification detection system comprises primers, probes, nicking endonucleases, DNA polymerases and ribonuclease endonucleases; the primers contain a nicking endonuclease cleavage recognition sequence; the probes contain at least one ribonucleotide base, and the ribonucleotide base can be complementary to the target sequence.

21. A use of a nickase and an endoribonuclease for preparing a highly sensitive nucleic acid isothermal amplification detection system, characterized in that: The nucleic acid isothermal amplification detection system comprises primers, probes, nicking endonucleases, DNA polymerases and ribonuclease endonucleases; the primers contain a nicking endonuclease cleavage recognition sequence; the probes contain at least one ribonucleotide base, and the ribonucleotide base can be complementary to the target sequence.

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