Nucleic acid isothermal amplification detection system and detection method thereof

Abstract

The invention provides a nucleic acid isothermal amplification detection system and a detection method thereof, which are characterized in that a primer containing a cleavage recognition sequence of incision endonuclease and a probe containing at least one ribonucleotide base are designed through combined use of incision endonuclease, DNA polymerase (reverse transcriptase is added for an RNA template) and ribonucleic acid endonuclease, and the nucleic acid isothermal amplification detection system is obtained. The primers and the probes are subjected to basic group modification, so that the signal intensity of the target nucleic acid is continuously amplified in the isothermal amplification process, the detection sensitivity is remarkable, meanwhile, mismatching can be avoided, non-characteristic amplification is reduced, the detection specificity is improved, missing detection and wrong detection are prevented, the detection time can be shortened, and the application range can be widened.

Description

Technical Field

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

[0002] Nucleic acid detection has been widely applied in the fields of infectious disease or genetic disease diagnosis, epidemiological investigation, tumor mutant gene detection, etc. Compared with immunological detection techniques, molecular diagnosis has higher sensitivity, and the results are more accurate and reliable. The most critical step in nucleic acid detection is nucleic acid amplification technology. Polymerase chain reaction (PCR) is a classical and mature in vitro nucleic acid amplification method. However, the application scenarios are still limited due to large and precise instruments and complex operation steps. In recent years, isothermal in vitro nucleic acid amplification technologies have 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-based amplification technology), RCA (rolling circle replication nucleic acid amplification technology), NEAR (nickase amplification reaction technology), etc.

[0003] Among them, NEAR (nickase amplification reaction technology) is to form a small nick on the DNA section, and then let the polymerase find that place to start replication, continuously forming nicks and continuously replicating. This is a very ingenious method that does not require PCR reaction and thermal cycling, making the nucleic acid amplification process very simple and faster.

[0004] With the discovery of single-strand nicking restriction endonucleases (also known as nickases), people gradually use them to replace double-strand restriction endonucleases and modified single nucleotide methods. In 2000, the scientific team of New England Biolabs, Inc. established an amplification reaction based on Nt.BstNBI nickase and Bst DNA polymerase, and respectively proved that other nickases such as Nt.Alw I or N.Mly I, etc. can also be applied to double-strand amplification (WO01 / 94544A2). The EXPAR (exponential amplification reaction) detection experiment of Van Ness J. et al. in 2003 with DNA polymerase and nickase as the amplification system had an extremely rapid amplification speed and could complete 10 within 5 minutes 6The amplification multiples mentioned above. However, Eric Tan and other team members (in 2008) found that non-specific amplification or false positives cannot be excluded during amplification, and through measures such as separating the components of the buffer and enzyme and mixing them after heating, the degree of non-specific amplification was controlled and reduced. Traci Kiesling et al. in 2007 used Nt.Alw I and Nt.BstNBI for amplification detection reactions with DNA polymerase and named it the NESA amplification technique. Jianwei Jeffery Li et al. in 2008 also carried out similar amplification detections using N.Bbvc IA, N.Bbvc IB and N.BstNBI.

[0005] In the 1960s, Kornberg proposed the possibility that DNA polymerase has the ability of DNA synthesis independent of primers or templates. With the continuous in-depth research, this ability of DNA polymerase was confirmed and called Ab initio, de novo synthesis (1997, 1998, Ogata et al.). In 2006, Xingguo Liang et al. found that when restriction endonucleases or nicking enzymes are present at the same time, it effectively promotes this non-template or primer-dependent synthesis. Subsequently, the research teams of Xingguo Liang and the Russian research team Nadezhda V. Zyrina et al. respectively studied the amplification models of de novo synthesis and confirmed that in the presence of nicking enzymes or restriction endonucleases, DNA polymerase can synthesize short nicking enzyme or restriction endonuclease recognition sequences or similar tandem repeat fragments, and then these fragments are rapidly amplified and extended to form a large number of non-specific Ab initio amplification products, which has become one of the important factors restricting the use of such enzymes for nucleic acid amplification.

[0006] Ribonuclease H (RNase H) is a functional enzyme widely present in prokaryotic and eukaryotic cells and participates in the excision repair process of genomic sequences. It can effectively degrade the RNA strand in the RNA-DNA hybrid strand and is one of the commonly used tool enzymes. Miller et al. first discovered RNase H in Escherichia coli in 1973, and then RNase H was cloned and localized. In 1990, Itaya, M. successfully proved the existence of a second RNase H (RNase H2) in Escherichia coli K-12, which also has the function of degrading RNA in the RNA-DNA hybrid strand. In 1999, Ohtani N. et al. identified a third RNase H (RNase H3) from Bacillus subtilis.

[0007] The inventor previously independently developed an isothermal amplification detection technology (CN202211195457). By utilizing the asterisk activity characteristic of nicking enzyme with mismatch cleavage, a primer-probe set with a nicking enzyme cleavage recognition sequence was designed to achieve a cyclic process in which a template strand continuously binds to the probe and is continuously cleaved to release fluorescence signals, generating stronger fluorescence signals and improving the detection sensitivity. Although this technical method can increase the fluorescence signal intensity and sensitivity of isothermal amplification detection, it requires the target sequence to contain a specific mismatch sequence, so this method has certain limitations in terms of the applicable scope.

[0008] Therefore, how to improve the nucleic acid isothermal amplification detection technology based on nicking enzyme, reduce non-specific 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 urgently needs to be solved at present. Summary of the Invention

[0009] To solve the above problems, the present invention provides a novel nucleic acid isothermal amplification detection system and its detection method. By combining the use of nicking endonuclease, DNA polymerase (reverse transcriptase is added for RNA templates), and ribonuclease, primers containing the cleavage recognition sequence of nicking endonuclease and probes containing at least one ribonucleotide base are designed, and the bases of the primers and probes are modified, so as to continuously amplify the signal intensity of the target nucleic acid during the isothermal amplification process, significantly improving the detection sensitivity, detection specificity, and accuracy. At the same time, the detection time is further shortened, the detection efficiency is improved, false negatives and false positives are prevented, and the applicable scope is broadened.

[0010] On the one hand, the present invention provides a nucleic acid isothermal amplification detection system, which includes primers, probes, nicking endonuclease, and ribonuclease; the primers contain the cleavage recognition sequence of nicking endonuclease; the probes contain at least one ribonucleotide base that can be complementary to the target sequence.

[0011] Furthermore, the system further includes DNA polymerase or reverse transcriptase; when the target sequence is a DNA sequence, DNA polymerase is also included, and when the target sequence is an RNA sequence, reverse transcriptase is also included.

[0012] The present invention provides a completely new nucleic acid (including DNA and RNA) isothermal amplification detection system based on nicking endonuclease, DNA polymerase (reverse transcriptase is added for RNA templates), and ribonuclease. Through the design of primers and probes and repeated cyclic amplification under the combined action of nicking endonuclease, ribonuclease, and nucleic acid polymerase, rapid nucleic acid amplification detection is achieved. The design of primers and probes mainly includes the following two aspects:

[0013] 1. By using the base sequence of the nicking enzyme on the complementary strand of the 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, and under the action of DNA polymerase, a cyclic process is realized in which the cut fragment continuously extends along the template, another template strand continuously binds to the primer and extends, and the amplicon is continuously cut and released.

[0014] 2. By using the property that ribonuclease can hydrolyze RNA in the DNA / RNA hybrid, a probe containing ribonucleotide bases is designed. During the isothermal amplification process, the ribonuclease cuts the probe, realizing a cyclic process in which a template strand continuously binds to the probe and is continuously cut and releases a fluorescence signal, effectively increasing the fluorescence intensity of the target nucleic acid to be detected, improving the detection sensitivity, and further shortening the detection time.

[0015] In the existing nicking enzyme isothermal amplification, only one fluorescence probe can bind to one template strand, and the fluorescence signal is relatively low. Especially in the initial stage of amplification, it is difficult to quickly obtain the detection result. For example, when using a molecular beacon probe, according to the luminescence mechanism of the molecular beacon fluorescence probe, before binding to the template strand, the molecular beacon fluorescence probe is in a U-shaped hairpin structure, and the fluorescence group and the quenching group located at both ends are close to each other and cannot emit fluorescence. When the fluorescence probe binds to the amplification product, the U-shaped hairpin structure opens into a straight chain, the fluorescence group and the quenching group move away from each other, and thus fluorescence is emitted. Moreover, as the amplification continues and the amplification product changes from single-stranded to double-stranded, the fluorescence probe will also fall off, causing the fluorescence group and the quenching group to approach each other again and the fluorescence to disappear again. Therefore, the nucleic acid detection sensitivity of the existing nicking enzyme isothermal amplification is relatively low, and accurate detection is difficult to achieve.

[0016] In the isothermal amplification using the probe (such as a fluorescence probe) provided by the present invention, since a ribonuclease recognition site is set on the fluorescence probe and the amplification product does not contain ribonucleotides and will not be cut by ribonuclease. Therefore, once the fluorescence probe binds to the single-stranded amplification product to form a double-stranded structure, it will be recognized and cut by ribonuclease. After being cut and detached, the probe will emit fluorescence (the fluorescence group and the quenching group of the probe are completely separated). New fluorescence probes continue to bind to the single-stranded amplification product, and so on. The probe is continuously cut and releases fluorescence signals, continuously amplifying the fluorescence signal of this template strand, and significantly improving the detection sensitivity.

[0017] Therefore, compared with the existing method of binding and releasing fluorescence signals by one probe and one template strand, the method provided by the present invention can effectively increase the fluorescence intensity and detection sensitivity of the target nucleic acid to be detected, and can also broaden the applicable range.

[0018] In existing nicking enzyme isothermal amplification, the problem of non-specific amplification may also occur, leading to missed detection and misdetection. Because there is a possibility that DNA polymerase has the ability of DNA synthesis independent of primers or templates, in the nicking enzyme isothermal amplification system, DNA polymerase can synthesize short nicking enzyme or ribonuclease recognition sequences or similar tandem repeat fragments, and then these fragments are rapidly amplified and extended, forming a large number of non-specific amplification products, thus resulting in missed detection and misdetection.

[0019] The present invention by using a combination of nicking enzyme, DNA polymerase, and ribonuclease also helps to prevent non-specific amplification and improve the specificity of the detection result. The reason may be that ribonuclease itself also has high recognition specificity, and binding to the corresponding probe containing ribonucleotide bases can further specifically identify whether the amplified product is the target sequence.

[0020] In some embodiments, the nucleic acid isothermal amplification detection system provided by the present invention includes the primer-probe set, and the primer-probe set includes a forward primer, a downstream primer, and a probe. The upstream primer and the downstream primer both contain the cleavage recognition sequence of the nicking enzyme. Since nicking enzyme isothermal amplification is carried out by the nicking enzyme cutting the primer to extend and amplify the template strand, both the upstream primer and the downstream primer must contain the cleavage recognition sequence.

[0021] When the target sequence is a DNA sequence, the nucleic acid isothermal amplification detection system further includes DNA polymerase; the temperature of the isothermal amplification is 50-60°C. The DNA polymerase (Bst DNA polymerase), nicking endonuclease Nt.BstNBI, and ribonuclease RNase H2 provided by the present invention can better play their roles at a temperature of 50-60°C, so as to carry out efficient nicking enzyme isothermal amplification and 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 needs 5-8 minutes.

[0022] 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.

[0023] Each nicking enzyme has its specific cleavage recognition sequence. When the single strand containing this cleavage recognition sequence binds to the complementary strand to form a double strand, the nicking enzyme can cleave the single strand containing the cleavage recognition sequence (that is, it must form a double strand before cleaving the single strand containing the recognition sequence), and the cleavage site is near the cleavage recognition sequence. The recognition sequences and cleavage positions of different nicking enzymes may be different. For example, Nt.BstNBI can cleave the single strand containing the recognition sequence at a position 4 nucleotides from the 3' end of the recognition sequence.

[0024] Theoretically, any nicking enzyme can be applied to the nucleic acid isothermal amplification detection system of the present invention. Since the cleavage recognition sequences of different nicking enzymes are also different, the cleavage recognition sequence of any nicking enzyme in the present invention is uniformly represented as "5'-NNNNNNNNNN-3'", where N is any one of the four nucleotide bases A, C, T, and G, and the number of Ns changes accordingly according to the recognition sequence of different nicking enzymes. However, the detection sensitivity, specificity, and accuracy of the systems constructed with different nicking enzymes may vary.

[0025] In some embodiments, the nicking enzyme is Nt.BstNBI, and its cleavage recognition sequence is "5'-GAGTC-3'".

[0026] In some embodiments, both the primers (including the upstream primer and the downstream primer) must contain "5'-GAGTC-3'".

[0027] In some embodiments, the recognition and cleavage sites of the nicking enzyme Nt.BstNBI are as follows:

[0028] 5'-N...NNN GAGTC NNNN...N-3'

[0029] 3'-N...NNN CTCAG NNNN...N-5'

[0030] Wherein, N = A, C, G or T, and the cleavage site sequence is 5'-GAGTC-3'.

[0031] Furthermore, the ribonuclease is RNase H2.

[0032] Three main types of RNaseH have now been found to exist in nature: RNaseH1, RNaseH2, and H3. RNase H1 is commonly used to destroy the RNA template after synthesizing complementary DNA (cDNA) by reverse transcription, and can also be used to cleave specific RNA sequences in the presence of short complementary fragments of DNA; RNase H2 can be used to degrade the RNA primer component of Okazaki fragments, or introduce single-strand cleavage at positions containing ribonucleotides. It has been reported that PCR experiments have been carried out using thermostable RNase H2; RNase H3 is more similar in structure to H2, but its activity is closer to that of H1.

[0033] RNA / DNA complexes have three forms: 1) single ribonucleotide RNA contained in double-stranded DNA; 2) composed of one RNA strand and one DNA strand; 3) RNA / DNA hybrid fragments of four or more consecutive ribonucleotides.

[0034] 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 types of RNA / DNA complexes above and exercise cleavage activity, but there may be a risk of off-target; some RNase H3 can also cleave the first type of complex, but with lower efficiency. RNase H2 nuclease can recognize the incorporation of a single ribonucleotide that appears in the DNA double strand and cleave at the 5' end of the ribonucleotide. Therefore, the ribonuclease in the nucleic acid isothermal amplification detection system of the present invention preferably selects RNase H2. It can be understood that ribonucleases with similar activities to RNase H2 can all be applied to construct the nucleic acid isothermal amplification detection system provided by the present invention.

[0035] Based on the recognition and cleavage activity of RNase H2, the present invention uses the reverse complementary sequence of the target sequence as the probe recognition sequence, and selects 1-30 deoxyribonucleotide bases on this sequence to be replaced with corresponding ribonucleotide bases. When the fluorescent probe is in the recognition process, it will complementarily pair and bind with the specifically amplified product in the amplicon. At the same time, because there are ribonucleotide bases on the probe, 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 this time, another probe can bind to the template. Therefore, a single template strand may bind multiple probes and the probes will be cleaved by RNase H2 to release fluorescent signals. This cycle repeats, enabling each template strand to produce a stronger fluorescent response.

[0036] The probe and isothermal amplification method provided by the present invention are suitable for RNase H2 from biological sources such as prokaryotes and eukaryotes. Since the cleavage sites of different RNase H2s are also different, the cleavage recognition sequence of any RNase H2 in the present invention is uniformly represented as "5'-rN-3'", where rN is any one of the four ribonucleotide bases A, C, U, and G, and the number of Ns changes accordingly according to the recognition sequence of different RNase H2s.

[0037] In the amplification system, the present invention introduces one or more RNase H2 cleavage recognition sites on the probe. The recognition site can be any one or a combination of ribonucleotide bases U, A, C, and G, which is uniformly represented as rN here. The sequence of the probe is "5'-N...NNNNrNNNNN...N-3'", where rN can be at any position in this sequence and can be one or more. When the fluorescent probe is in the recognition process, it will bind to the specifically amplified product in a complementary pairing manner. At the same time, because there are ribonucleotide bases on the probe, the probe bound to the target will be recognized and cleaved by RNase H2 to generate a fluorescent signal.

[0038] Further, the primer sequentially includes a stable 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 Taqman probe, MGB probe, and molecular beacon probe; the label on the probe is any one of fluorescein, biotin, digoxin, and isotope.

[0039] Research has proven that RNase H2 nuclease has good cleavage activity in the nucleic acid isothermal amplification detection system provided by the present invention. The probe containing ribonucleotide bases can bind to single-stranded template DNA to form a double-strand and be cleaved by RNase H2 enzyme to generate a strong fluorescent signal; when there is no template matching the probe in the reaction system, RNase H2 enzyme cannot cleave.

[0040] It can be understood that the present invention is applicable to any kind of probe. Research has proven that through the probe design method provided by the present invention, the detection sensitivity of nucleic acids can be significantly improved.

[0041] Further, the probe type is a molecular beacon probe.

[0042] Further, the label on the probe is fluorescein, and the two ends of the probe are respectively modified with a fluorescent group or a quenching group.

[0043] When one end of the fluorescent probe is modified with a fluorescent group, the other end is modified with a quenching group; or when one end is modified with a quenching group, the other end is modified with a fluorescent group.

[0044] In some embodiments, the fluorescent group is one or more of fluorescent groups such as FAM, TET, VIC, HEX, TAMRA, ROX, Cy5, LCRED640, etc.; the quenching group is one or more of quenching groups such as Dabcyl, BHQ1, BHQ2, BHQ3, MGB, etc.

[0045] Furthermore, the primer and / or probe contains base modifications, and the base modifications include any one or more of 2'-O-methyl, thiol modification, alkyl, amino, deoxyinosine modification.

[0046] In the isothermal amplification detection system provided by the present invention, when using nicking endonuclease, ribonuclease and DNA polymerase in combination, there will still be a problem of non-specific amplification, resulting in missed detection and misdetection. Because DNA polymerase has the possibility of DNA synthesis ability independent of primers or templates, when ribonuclease or nicking enzyme exists simultaneously, it will effectively promote the occurrence of this non-template or primer-dependent synthesis. That is to say, in the presence of nicking enzyme or ribonuclease, DNA polymerase can synthesize short nicking enzyme or ribonuclease recognition sequences or similar tandem repeat fragments, and then these fragments are rapidly amplified and extended to form a large number of non-specific amplification products, thus leading to missed detection and misdetection.

[0047] The present invention helps to avoid mismatching to reduce non-specific amplification and improve the accuracy and specificity of detection results by introducing specific base modifications into primers and probes.

[0048] Furthermore, the base modification is 2'-O-methyl modification, and the bases at the 3' end of the primer and / or the bases upstream and downstream of the ribonucleic acid base site of the probe contain base modifications.

[0049] Research has shown that compared with other base modification methods, 2'-O-methyl modification has a more significant effect in improving the accuracy and specificity of detection results.

[0050] Since the amplification and extension of the primer start from the 3'' end, it is necessary to modify the bases at the 3' end of the primer.

[0051] Since the probe used is a molecular beacon probe containing ribonucleic acid bases, the probe recognizes the complementary strand of the target sequence of the amplification product and binds to it to form a DNA / RNA hybrid strand. At the same time, the ribonucleic acid base probe of the hybrid strand is cut under the action of ribonuclease to emit fluorescence. Therefore, it is necessary to modify the bases upstream and downstream of the ribonucleic acid base site of the probe to avoid mismatching.

[0052] In primers and / or probes, the number of bases that specifically need to be base-modified needs to be determined through specific experimental procedures. Since the number of bases that need to be base-modified may vary for primers or probes with different sequences, it needs to be determined through experimental screening.

[0053] Furthermore, bases at some specific positions of the primers and probes all contain base modifications.

[0054] Research has shown that 2'-O-methyl modification of primers and probes does not affect the cleavage activities of nicking enzymes and ribonucleases. Moreover, compared with the situation of only performing 2'-O-methyl modification on primers or only on probes, simultaneously performing 2'-O-methyl modification on primers and probes can more effectively prevent mismatching, prevent non-specific amplification, and improve amplification efficiency.

[0055] In some embodiments, 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 ribonucleic acid bases of the probe contain 2'-O-methyl modification.

[0056] In some embodiments, the present invention provides a nucleic acid isothermal amplification detection system for Mycoplasma pneumoniae, which includes a primer-probe group, a nicking endonuclease, a DNA polymerase, and a ribonuclease. The primer-probe group includes:

[0057] (1) MMP-16S-F (upstream primer): 5'-CGAACGGGT GAGTC ACACGTATCCAATCTG-3' (SEQ ID NO.1);

[0058] (2) MMP-16S-R (downstream primer): 5'-CTCCCGTAG GAGTC TGGGCCGTGTCTCAG-3' (SEQ ID NO.2);

[0059] (3) Probe MMP-BPr: 5'-ROX-GTCATC ACTA-rG-CTAATACC GATGAC-BHQ2-3' (SEQ ID NO.3).

[0060] The probe type is a molecular beacon probe, and both ends of the probe are modified with a fluorescent group or a quenching group; the ribonuclease is RNase H2 enzyme.

[0061] The corresponding template target nucleic acid sequence recognized by the probe MMP-BPr is 5’-NNNNN GGTATTAGCTAGTFor NNNNN-3', the probe can complementarily pair with the template. After the RNase H2 enzyme recognizes and cleaves the rG ribonucleotide base on the probe, the template target sequence will not be cleaved.

[0062] This system can be used for the highly sensitive detection of the 16S sequence of Mycoplasma pneumoniae.

[0063] Furthermore, the primers and probes of the 16S gene detection system for Mycoplasma pneumoniae are modified with 2'-O-methyl-, which does not affect the function of the probe and the detection of Mycoplasma pneumoniae. The sequences of the modified primers and probes are as follows:

[0064] MMP-16S-mF: 5'-CGAACGGGTGAGTCACACGmTmAmTmCmCmAmAmTCTG-3' (SEQ ID NO.4);

[0065] MMP-16S-mR: 5'-CTCCCGTAGGAGTCTGGGCmCmGmTmGmTmCmTmCAG-3' (SEQ ID NO.5);

[0066] MMP-16S-mBPr: 5'-ROX-GTCATCmAmCmTA-rG-CmTmAmATACCGATGAC-BHQ2-3' (SEQ ID NO.6) (where the m prefix represents that the base is modified with 2'-O-methyl-).

[0067] This system can be used for the highly sensitive, highly accurate, and highly specific detection of the 16S sequence of Mycoplasma pneumoniae.

[0068] In some embodiments, the present invention provides a nucleic acid isothermal amplification detection system for novel coronavirus, comprising a primer-probe set, a nicking endonuclease, a DNA polymerase, and a ribonuclease. The primer-probe set includes:

[0069] (1) COV-S-F (forward primer): 5'-ACCATACA GAGTC GAGTAGTACTTTCTTTTG-3' (SEQ ID NO.7);

[0070] (2) COV-S-R (reverse primer): 5'-CCAAGTAG GAGTC AGTTGATCTGCATGA-3' (SEQ ID NO.8);

[0071] (3) Probe COV-S-BPr: 5'-FAM-GGTGCAT-rA-TGTCAATTTCAACTGCACC-BHQ1-3' (SEQ ID NO.9).

[0072] This system can be used for the highly sensitive detection of the S gene of the novel coronavirus.

[0073] This system can be used for the highly sensitive, highly accurate, and highly specific detection of the S gene of the novel coronavirus.

[0074] On the other hand, the present invention provides a nucleic acid isothermal amplification detection method, which uses the nucleic acid isothermal amplification detection system described above for nucleic acid isothermal amplification detection.

[0075] On yet another aspect, 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 detecting a target sequence. The nucleic acid isothermal amplification detection system includes primers, probes, nicking endonucleases, DNA polymerases, and ribonucleases; the primers contain the cleavage recognition sequence of the nicking endonuclease; the probes contain at least one ribonucleotide base that can be complementary to the target sequence.

[0076] On yet another aspect, the present invention provides the use of a nicking enzyme for preparing a reagent for improving the detection sensitivity of a nucleic acid isothermal amplification detection system. The nucleic acid isothermal amplification detection system includes primers, probes, nicking endonucleases, DNA polymerases, and ribonucleases; the primers contain the cleavage recognition sequence of the nicking endonuclease; the probes contain at least one ribonucleotide base that can be complementary to the target sequence.

[0077] On yet another aspect, the present invention provides the use of a probe for preparing a reagent for improving the detection sensitivity of a nucleic acid isothermal amplification detection system. The nucleic acid isothermal amplification detection system includes primers, probes, nicking endonucleases, DNA polymerases, and ribonucleases; the primers contain the cleavage recognition sequence of the nicking endonuclease; the probes contain at least one ribonucleotide base that can be complementary to the target sequence.

[0078] On yet another aspect, the present invention provides the use of a nicking enzyme and a ribonuclease for preparing a highly sensitive nucleic acid isothermal amplification detection system. The nucleic acid isothermal amplification detection system includes primers, probes, nicking endonucleases, DNA polymerases, and ribonucleases; the primers contain the cleavage recognition sequence of the nicking endonuclease; the probes contain at least one ribonucleotide base that can be complementary to the target sequence.

[0079] The nucleic acid isothermal amplification detection system provided by the present invention has the following beneficial effects:

[0080] 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.

[0081] 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;

[0082] 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.

[0083] 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;

[0084] 5. The application of this nucleic acid isothermal amplification detection system in POCT products has huge market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 Schematic diagram of primer design principle;

[0086] Figure 2 Schematic diagram of the probe design principle;

[0087] Figure 3 Schematic diagram of the detection results of different probe sequences in Example 1;

[0088] Figure 4 This is a schematic diagram of the sensitivity results of the novel coronavirus nucleic acid detection in Example 2;

[0089] Figure 5 Schematic diagram of the test results of different primer probes used in Example 3 for Mycoplasma pneumoniae samples with different concentrations;

[0090] Figure 6 Schematic diagram of the specific results of the circulating probe cleavage amplification nucleic acid detection of Mycoplasma pneumoniae in Example 3. DETAILED DESCRIPTION

[0091] The preferred embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate the 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 are obtained by purchasing commercially available products.

[0092] In the following examples, the nicking enzyme used is Nt.BstNBI nicking enzyme purchased from NEB, product catalog number #R0607S, the DNA polymerase used is Bst DNA polymerase purchased from Thermo Fisher Scientific Inc., product catalog number A56656, the RNase H2 enzyme is purchased from Integrated DNA Technologies, Inc., product catalog number #11-03-02-03, and the RTx reverse transcriptase is purchased from NEB, product catalog number #M0380L.

[0093] Example 1: Verification of the recognition and cleavage activity of RNase H2 enzyme

[0094] Taking RNase H2 as an example, this example verified the cleavage characteristics of RNase H2 through experiments, that is, when a probe with ribonucleotide bases forms a DNA / RNA hybrid strand with the target, the RNase H2 enzyme will cleave it, but when the probe with ribonucleotide bases does not recognize the nucleic acid complementary to it, the RNase H2 enzyme does not have a cleavage effect.

[0095] According to the RNase H2 enzyme cleavage recognition sequence (5'-NNNNN-rN-NNNN-3', N represents any deoxyribonucleotide base, rN represents any ribonucleotide base) and the molecular beacon probe design rules, two probes containing different numbers of ribonucleotides were designed. The schematic diagram of the design principle is shown in Figure 2 . Fluorescent group FAM and quenching group BHQ1 were respectively labeled at the 5' and 3' of the probe sequence. Specifically:

[0096] ASV-NN-R3Probe1:

[0097] 5'-FAM-TGTGTTACTTT GAGTrC AAATCGAAGAAACACA-BHQ1-3' (SEQ ID NO.10);

[0098] ASV-NN-R3Probe2:

[0099] 5'-FAM-TGTGTTACTTT GAGrUrCr ArArArUrCrGAAGAAACACA-BHQ1-3' (SEQ ID NO.11);

[0100] The target recognition sequence corresponding to this probe is:

[0101] ASV-TR7: 5'-TGCGTTTCTTCGATTT- GACTC- AAAGTAAACAG-3' (single-stranded template). (SEQ ID NO. 12).

[0102] Synthesize the above probe and single-stranded template DNA fragment, and perform a cleavage reaction using RNase H2 enzyme. The specific reaction system is shown in Table 1.

[0103] Table 1. RNase H2 enzyme digestion reaction system and components

[0104] Component Volume (μL) Single-stranded template fragment (10 μM) 2 μL Probe (10 μM) (in excess) 1 μL Reaction buffer (10×) 2 μL RNase H2 enzyme 20U Sterile water 14 μL Total volume 20 μL

[0105] The experimental design is as follows: 1: Use ASV-NN-R3Probe1 probe and ASV-TR7 as the template in the reaction system; 2: Use ASV-NN-R3Probe2 probe and ASV-TR7 as the template in the reaction system; 3: Use ASV-NN-R3Probe1 probe and water as the template in the reaction system; 4: Use ASV-NN-R3Probe2 probe and water as the template in the reaction system.

[0106] Use a fluorescence isothermal amplification instrument (GS8) to perform the reaction and real-time detection. The reaction conditions are 60 °C for 30 minutes, and the fluorescence acquisition period is 30 seconds / point.

[0107] The experimental results are as Figure 3 shown. 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 cleaved by RNase H2 enzyme, generating a strong fluorescence signal ( Figure 3 1 and 2 in); when there is no template matching the probe in the reaction system, RNase H2 enzyme cannot cleave ( Figure 3 3 and 4 in).

[0108] It should be noted that in addition to RNase H2, other nucleases, etc. have similar cleavage characteristics. Probes containing the cleavage sites of these nucleases can also achieve this function and all fall within the protection scope of this application.

[0109] Example 2. Detection of novel coronavirus

[0110] According to the primer and probe design principles of the nucleic acid isothermal amplification detection technology of the present invention, the S gene sequence of the novel coronavirus, and the characteristics of Nt.BstNBI, Nt.BspD6I nicking enzymes and RNase H2 enzyme, specific primers and probes for detecting the S gene of the novel coronavirus are designed and synthesized. The design rules of the probes are asFigure 2 As shown, it includes an identification region capable of binding to a target strand and an RNase H2 enzyme recognition site. During the nicking enzyme isothermal amplification process, the primer rapidly amplifies the sequence of the detection region under the action of the nicking enzyme and polymerase. After the probe recognizes the target sequence and binds to form a double strand, the RNase H2 enzyme hydrolyzes the RNA bases in the substrate of the hybrid strand. Under the combined action of the nicking endonuclease, ribonuclease, and nucleic acid polymerase, rapid nucleic acid amplification detection is achieved. In this example, by screening a suitable target segment, multiple pairs of primer-probes were designed. The identification regions, nicking enzyme recognition sites, and stable regions in different primers are different. After multiple screenings, the following optimal primer sequences (forward primer and reverse primer) were finally obtained, and the influence of different probe sequences on the nucleic acid detection results was investigated, as follows:

[0111] COV-S-F: 5'-ACCATACA GAGTC GAGTAGTACTTTCTTTTG-3' (SEQ ID NO.7)

[0112] COV-S-R: 5'-CCAAGTAG GAGTC AGTTGATCTGCATGA-3' (SEQ ID NO.8)

[0113] COV-S-BPr: 5'-FAM-GGTGCAT-rA-TGTCAATTTCAACTGCACC-BHQ1-3' (SEQ ID NO.9)

[0114] Using the purchased RNA reference material of the novel coronavirus S gene (number: NIM-RM5208, National Institute of Metrology, China) as a template for testing and detection. The specific implementation steps are as follows:

[0115] Step 1: In an 8-tube PCR reaction tube, prepare an isothermal amplification detection system according to Table 2.

[0116] Table 2. Isothermal amplification detection system for novel coronavirus nucleic acid

[0117]

[0118]

[0119] Step 2: Use TE Buffer to serially dilute the RNA reference material of the novel coronavirus S gene to 200 copies / μl, 40 copies / μl, 20 copies / μl, 10 copies / μl, 5 copies / μl, 2 copies / μl, 1 copy / μl.

[0120] Step 3: Add 5 μl of the diluted templates of the novel coronavirus S gene RNA standard substance with different concentrations to the 8-well PCR reaction tubes prepared in Step 1;

[0121] Step 4: After shaking and mixing the 8-well PCR reaction tubes containing the complete reagent system, place them in a fluorescence constant temperature amplification instrument (GS8) for reaction and real-time detection. The reaction conditions are 50 °C for 10 minutes, and the fluorescence acquisition period is 15 seconds per point.

[0122] 2. Experimental results

[0123] The experimental results are as Figure 4 shown. The copy numbers of samples 1 - 8 are 1000 copies, 200 copies, 100 copies, 50 copies, 25 copies, 10 copies, 5 copies, and negative respectively. From the Figure 4 results, it can be seen that when the novel coronavirus S gene RNA standard substance is at 5 - 1000 copies, the isothermal amplification reaction has a high fluorescence response, indicating that the method in this example can detect the novel coronavirus S gene RNA standard substance. Since the novel coronavirus S gene RNA standard substance is RNA, therefore, this application is based on this nucleic acid isothermal amplification detection technology to amplify and detect the novel coronavirus S gene RNA, and the detection sensitivity of the novel coronavirus S gene RNA reaches 1000 copies / ml (converted according to the RNA template loading amount of 5 μl at 1 copy / μl, that is, 1000 copies / 1 ml).

[0124] Example 3. Application of the nucleic acid isothermal amplification detection system in the detection of Mycoplasma pneumoniae nucleic acid

[0125] In this example, for Mycoplasma pneumoniae, the following four forms of detection systems were investigated:

[0126] 1. The existing nicking enzyme amplification detection system (nicking enzyme and DNA polymerase), the primers contain the cleavage recognition sequence of the nicking endonuclease, but there is no RNase H2, and the probe has no RNase H2 cleavage site;

[0127] 2. The existing PCR detection system (DNA polymerase, without nicking enzyme), the primers contain the cleavage recognition sequence of the nicking endonuclease, but there is no RNase H2, and the probe has no RNase H2 cleavage site;

[0128] 3. Nicking enzyme, DNA polymerase and RNase H2, the primers contain the cleavage recognition sequence of the nicking endonuclease, and the probe contains the RNase H2 cleavage site;

[0129] 4. Nicking enzyme, DNA polymerase and RNase H2. The primer contains the cleavage recognition sequence of the nicking endonuclease, and the probe contains the cleavage site of RNase H2. At the same time, both the primer and the probe are 2'-O-methyl modified;

[0130] The experimental method is as follows:

[0131] In this example, by screening suitable target segments, multiple pairs of primer-probes were designed. The recognition regions, nicking enzyme recognition sites, and stable regions in different primers are different. After multiple screenings, the following optimal primer sequences (forward primer and reverse primer) were finally obtained. Then, according to the primer-probe design principle of the loop-mediated isothermal amplification detection reaction technology and the nucleic acid sequence of Mycoplasma pneumoniae, four different forms of Mycoplasma pneumoniae nucleic acid detection primers and probes were designed and synthesized, and then nucleic acid isothermal amplification detection was carried out. The primer-probes of the four detection systems are as follows:

[0132] The first type:

[0133] MMP-16S-F: 5'-CGAACGGGT GAGTC ACACGTATCCAATCTG-3' (SEQ ID NO.1);

[0134] MMP-16S-R: 5'-CTCCCGTAG GAGTC TGGGCCGTGTCTCAG-3' (SEQ ID NO.2);

[0135] MMP-16S-BP0: 5'-ROX-GTCATCACTAGCTAATACCGATGAC-BHQ2-3' (SEQ ID NO.13);

[0136] The second type:

[0137] MMP-16S-F0: 5'-CGAACGGGTGAGTAACACGTATCCAATCTG-3' (SEQ ID NO.22);

[0138] MMP-16S-R0: 5'-CTCCCGTAGGAGTATGGGCCGTGTCTCAG-3' (SEQ ID NO.23);

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

[0140] The third type:

[0141] MMP-16S-F: 5'-CGAACGGGT GAGTC ACACGTATCCAATCTG-3'(SEQ ID NO.1);

[0142] MMP-16S-R: 5'-CTCCCGTAG GAGTC TGGGCCGTGTCTCAG-3'(SEQ ID NO.2);

[0143] MMP-16S-BPr: 5'-ROX-GTCATCACTA-rG-CTAATACCGATGAC-BHQ2-3'(SEQ ID NO.3);

[0144] The fourth type:

[0145] MMP-16S-mF: 5'-CGAACGGGTGAGTCACACGmTmAmTmCmCmAmAmTCTG-3'(SEQ ID NO.4);

[0146] MMP-16S-mR: 5'-CTCCCGTAGGAGTCTGGGCmCmGmTmGmTmCmTmCAG-3'(SEQ ID NO.5);

[0147] MMP-16S-mBPr: 5'-ROX-GTCATCmAmCmTA-rG-CmTmAmATACCGATGAC-BHQ2-3'(SEQ ID NO.6);

[0148] Using inactivated Mycoplasma pneumoniae culture (1×10 9 CCU / mL, No.: 20210603, prepared by Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences) as a template for the cycle amplification detection reaction. The specific experimental steps are as follows:

[0149] Step 1: In the PCR reaction tube, prepare the cycle amplification detection system according to Table 2;

[0150] Step 2: Use TE Buffer to serially dilute the inactivated Mycoplasma pneumoniae culture to 5000 CCU / mL, 500 CCU / mL, and 50 CCU / mL;

[0151] Step 3: Take 1 mL of inactivated Mycoplasma pneumoniae culture with different concentrations respectively, and perform nucleic acid extraction using a DNA / RNA nucleic acid extraction kit (TianGen, DF438-01), and dissolve it in 30 μL of TE solution;

[0152] Step 4: Add 5 μL of the nucleic acid extracted in Step 3 to the PCR reaction tube prepared in Step 1;

[0153] Step 5: After shaking and mixing the PCR reaction tube containing the complete reagent system, place it into a real-time fluorescence quantitative PCR instrument (Thermofisher, QuantStudio 3) for reaction and real-time detection. The reaction conditions are: 55°C, duration of 20 minutes, fluorescence acquisition cycle of 30 seconds per point, and directly perform isothermal nucleic acid amplification detection reaction.

[0154] Table 3. Reagent system for detecting Mycoplasma pneumoniae by amplification detection method

[0155]

[0156]

[0157] Examine the detection sensitivities of four forms of detection systems. The experimental results are as Figure 5 shown.

[0158] From Figure 5 the results in it, it can be seen that: compared with using the MMP-16S-BP0 probe, when using the MMP-16S-BPr probe, the fluorescence response value of the nicking enzyme polymerase isothermal amplification reaction increases significantly, indicating that when using the MMP-16S-BPr probe, the sensitivity of the nicking enzyme polymerase isothermal amplification reaction is significantly improved (the earlier the peak starting time, the higher the fluorescence response value, and the higher the sensitivity). When using the primer-probe modified with 2'-O-methyl for nucleic acid isothermal amplification detection reaction, it still has a high fluorescence intensity and can perform high-sensitivity detection of Mycoplasma pneumoniae.

[0159] By comparing the four forms of primer-probes, it can be found that compared with the first and second forms, the probe of MMP-16S-BPr (the third detection system) or MMP-16S-mBPr (the fourth detection system) has a ribonucleoside base in the sequence "5'-NNNrNNNN-3'", and after complementary pairing with the target sequence to form a double strand, it will be recognized and cleaved by the RNase H2 enzyme, indicating that using a probe containing the RNase H2 enzyme recognition site can improve the sensitivity of the cyclic amplification detection reaction.

[0160] At the same time, by comparing the four forms of primer-probes, it can be found that on the premise of the same sample to be detected, the positive peak starting time of the MMP-16S-BPr or MMP-16S-mBPr probe is much earlier than that of MMP-16S-BP0, indicating that using a probe containing the RNase H2 enzyme recognition site can significantly improve the detection speed of the cyclic amplification detection reaction.

[0161] From Figure 5It can be seen that the use of the fluorescent probes MMP-16S-BPr or MMP-16S-mBPr containing the cleavage recognition site of RNase H2 enzyme can significantly increase the fluorescence value of the nucleic acid detection result and improve the detection sensitivity, that is, both detection system three and detection system four can detect Mycoplasma pneumoniae samples at 50 CCU / mL. Detection system one and detection system two can respectively detect Mycoplasma pneumoniae samples at a minimum of 500 CCU / mL and 5000 CCU / mL. See Table 4 for details.

[0162] Table 4 Sensitivity and corresponding average peak appearance time of different detection systems

[0163] Detection system Detection system 1 Detection system 2 Detection system 3 Detection system 4 Sensitivity 500 CCU / mL 5000 CCU / mL 50 CCU / mL 50 CCU / mL Average peak time 6.5 min 18 min 5.5 min 5.5 min

[0164] Through the above analysis, it can be concluded that: compared with the first detection system, the detection sensitivity, positive peak appearance speed and detection fluorescence intensity of the third and fourth detection systems with the addition of RNase H2 enzyme and the probe combination containing the corresponding recognition site have been significantly improved, indicating that when using RNase H2 enzyme and MMP-16S-BPr probe, the detection sensitivity, detection efficiency and signal-to-noise ratio of the nucleic acid detection system can be significantly improved; compared with the second detection system, the detection sensitivity, positive peak appearance speed and detection fluorescence intensity of the third and fourth detection systems with the addition of Nt.BstNBI enzyme and the primer combination containing the corresponding recognition site have been significantly improved, indicating that when using nicking enzyme and the primer combination containing the corresponding recognition site, the detection sensitivity, detection efficiency and signal-to-noise ratio of the nucleic acid detection system can be significantly improved; compared with the third detection system, the primer-probe combination with methylation modification in the fourth detection system can also obtain comparable detection sensitivity and positive peak appearance speed, and in addition, the fluorescence intensity is also improved to a certain extent, indicating that the primer-probe combination with methylation modification will not affect the detection effect of the third detection system.

[0165] Examine the negative coincidence rate of the four forms of detection systems. Ten throat swab samples from healthy people were respectively subjected to nucleic acid extraction using a DNA / RNA nucleic acid extraction kit (TianGen, DF438-01), dissolved in 30 μL of TE solution, and used as negative samples to test the negative coincidence rate of the four forms of detection systems. The detection results are shown in Table 5:

[0166] Table 5 Negative detection results of different detection systems

[0167]

[0168] As can be seen from Table 5, the negative coincidence rates of 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 compliance (i.e., false positive phenomenon), the first and second detection systems are inferior to the third and fourth detection systems, and misdetection may occur. The reason may be that the RNase H2 enzyme can further improve the recognition specificity.

[0169] To investigate the detection specificity of the fourth detection system, 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 were used as templates. The amplification was carried out using the three detection systems described above. Among them, the inactivated cross-experiment 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, the inactivated strains of H5N1 and H3N2 were purchased from Shenzhen Haisen Biotechnology Co., Ltd., and the inactivated strains of Ureaplasma urealyticum and Mycoplasma hominis were purchased from Guangzhou Bangdesheng Biotechnology Co., Ltd.

[0170] Among them, the detection results of the fourth detection system are as Figure 6 shown. Sample 19 is the nucleic acid of Mycoplasma pneumoniae, and samples 1-18 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. From Figure 6 the results in it, it can be seen that only the amplification detection reaction with the nucleic acid of Mycoplasma pneumoniae as the template can produce a fluorescence response, and there is no fluorescence response or the response is almost 0 when other bacteria or viruses such as Klebsiella pneumoniae, Streptococcus pneumoniae, and Ureaplasma urealyticum are used as templates. This result shows that the nucleic acid amplification detection reaction of the present invention has good specificity and the detection results are very accurate.

[0171] In this example, it is further proved through research that for some complex viruses, the fourth form of detection system helps to better prevent non-specific amplification, improve the specificity of detection results, and thus prevent missed and misdetected cases. For example, when detecting African swine fever virus, if the primer-probe group is not modified with 2'-O-methyl, non-specific amplification still exists. However, after the primer-probe group is modified with 2'-O-methyl, the problem of non-specific amplification can be completely solved, thereby improving the specificity of the detection results.

[0172] Example 4. Influence of Different Nicking Enzymes on the Nucleic Acid Detection of Mycoplasma pneumoniae

[0173] In this example, the method provided in Example 3 is adopted, and the fourth form of primer-probe group is used for nucleic acid amplification. The nicking enzymes are replaced with the following several nicking enzymes: Nb.BsmI (recognition sequence 5'-GAATCG-3’), Nb.BsrDI (recognition sequence 5'-GCAATG-3’), Nt.BspQI (recognition sequence 5'-GCTCTTC-3’), Nt.AlwI (recognition sequence 5'-GGTAC-3’), and the corresponding nicking enzyme recognition sequences are replaced in the upstream primer and the downstream primer respectively. The influence of using different nicking enzymes on the nucleic acid isothermal amplification detection system provided by the present invention is investigated.

[0174] The primer-probe sequence information is as follows:

[0175] Nt.BstNBI:

[0176] MMP-16S-mF: 5'-CGAACGGGT GAGTC ACACGmTmAmTmCmCmAmAmTCTG-3'(SEQ ID NO.4);

[0177] MMP-16S-mR: 5'-CTCCCGTAG GAGTC TGGGCmCmGmTmGmTmCmTmCAG-3'(SEQ ID NO.5);

[0178] MMP-16S-mBPr: 5'-ROX-GTCATCmAmCmTA-rG-CmTmAmATACCGATGAC-BHQ2-3'(SEQ IDNO.6);

[0179] Nb.BsmI:

[0180] MMP-16S-mF2: 5'-CGAACGGG GAATCG ACACGmTmAmTmCmCmAmAmTCTG-3'(SEQ IDNO.14);

[0181] MMP-16S-mR2: 5'-CTCCCGTA GAATCG TGGGCmCmGmTmGmTmCmTmCAG-3'(SEQ ID NO.15);

[0182] Nb.BsrDI:

[0183] MMP-16S-mF3: 5'-CGAACGGG GCAATG ACACGmTmAmTmCmCmAmAmTCTG-3'(SEQ ID NO.16);

[0184] MMP-16S-mR3: 5'-CTCCCGTA GCAATG TGGGCmCmGmTmGmTmCmTmCAG-3'(SEQ ID NO.17);

[0185] Nt.BspQI:

[0186] MMP-16S-mF4: 5'-CGAACGG GCTCTTC ACACGmTmAmTmCmCmAmAmTCTG-3'(SEQ ID NO.18);

[0187] MMP-16S-mR4: 5'-CTCCCGT GCTCTTC TGGGCmCmGmTmGmTmCmTmCAG-3'SEQ ID NO.19);

[0188] Nt.AlwI:

[0189] MMP-16S-mF5: 5'-CGAACGGGT GGTAC ACACGmTmAmTmCmCmAmAmTCTG-3'(SEQ ID NO.20);

[0190] MMP-16S-mR5: 5'-CTCCCGTAG GGTAC TGGGCmCmGmTmGmTmCmTmCAG-3'(SEQ ID NO.21);

[0191] The detection results are shown in Table 6.

[0192] Table 6. Influence of Different Kinds of Nicking Enzymes on Detection Results

[0193]

[0194] As can be seen from Table 6, different nicking enzymes can basically be applied to the nucleic acid isothermal amplification provided by the present invention. However, for different nicking enzymes, their peak starting times are different, and the detection speeds are different (the earlier the peak starting time, the faster the detection speed), and there may be differences in detection sensitivity. The most preferred nicking enzyme is Nt.BstNBI.

[0195] Research has proved that the nicking enzyme Nt.BstNBI is particularly suitable for the nucleic acid isothermal amplification detection system provided by the present invention. The main reason is that in the reaction conditions of this amplification detection system, the optimal reaction temperature of Nt.BstNBI is most suitable for the reaction temperature of this system, and it shows the best enzyme activity level under the reaction conditions of this detection. The 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 shown. According to the existing public information, the main reason is that the optimal reaction temperatures and enzyme activity efficiencies of different nicking enzymes are different.

[0196] In addition, this embodiment also designed corresponding primers and probes for the African swine fever B646L gene for nucleic acid isothermal amplification detection reaction, which also has very good detection sensitivity and specificity, and the detection sensitivity reaches 500 copies / ml.

[0197] The above results show that a method for improving the primer specificity in the recombinase activity reaction provided by the present invention can significantly improve the detection efficiency and detection specificity of the detection system and obtain higher detection sensitivity.

[0198] Although the above has been described with general descriptions and preferred embodiments, it is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An isothermal nucleic acid amplification detection system, characterized in that, It includes primers, probes, nicking endonucleases and ribonucleases; the primers contain the cleavage recognition sequences of nicking endonucleases; the probes contain at least one ribonucleotide base that can be complementary to the target sequence.

2. The nucleic acid isothermal amplification detection system according to claim 1, characterized in that, 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, in addition to DNA polymerase, it also includes reverse transcriptase.

3. The nucleic acid isothermal amplification detection system according to claim 2, wherein The nicking endonuclease includes Nt. Any one or more of 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.

4. The nucleic acid isothermal amplification detection system according to claim 3, wherein The ribonuclease is RNaseH2.

5. The nucleic acid isothermal amplification detection system according to claim 4, wherein The primers sequentially contain a stable 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 Taqman probes, MGB probes, and molecular beacon probes; the label on the probe is any one of fluorescein, biotin, digoxin, and isotopes.

6. The nucleic acid isothermal amplification detection system according to claim 5, wherein The probe type is a molecular beacon probe.

7. The nucleic acid isothermal amplification detection system according to claim 6, wherein The primers and / or probes contain base modifications, and the base modifications include any one or more of 2'-O-methyl, thiol modification, alkyl, amino, and deoxyinosine modification.

8. The nucleic acid isothermal amplification detection system according to claim 7, wherein The base modification is 2'-O-methyl modification, and the base at the 3'-end of the primers and / or probes contains the base modification.

9. 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 8. 10.2'-O-methyl modification is used for preparing a reagent for improving the accuracy and specificity of a nucleic acid isothermal amplification detection system for detecting a target sequence, characterized in that, The nucleic acid isothermal amplification detection system includes primers, probes, nicking endonucleases, DNA polymerase and ribonucleases; the primers contain the cleavage recognition sequences of nicking endonucleases; the probes contain at least one ribonucleotide base that can be complementary to the target sequence.

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