Method for detecting target nucleic acid to be detected by using melting curve and kit therefor

Abstract

The present invention provides a method and a kit for detecting a target nucleic acid to be detected by using a melting curve. The method includes designing a first primer, a second primer and a detection probe for the target nucleic acid sequence to be detected, performing PCR amplification in a PCR amplification system containing the first primer, the second primer, the detection probe, the sample to be detected, a nicking enzyme and a DNA polymerase, generating a reporter primer that is complementary to the detection probe to form a double-stranded product, obtaining the melting curve of the double-stranded product, and the melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be detected. The method of the present invention is a non-target-dependent melting curve method, and the melting point (T m value) of each double-stranded body can be calculated in advance, solving the problems of melting curve peak shift and easy misjudgment caused by the easy mutation of the target nucleic acid sequence to be detected.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular, to a method for detecting a target nucleic acid to be detected using a melting curve and a kit therefor. Background Art

[0002] Nucleic acid detection plays an increasingly important role in aspects such as molecular diagnosis, molecular biology research, animal and plant quarantine, and food safety detection. Currently, common nucleic acid detection methods mainly include real-time fluorescence PCR method, high-throughput sequencing method, microarray chip, nucleic acid invasion detection method, and multi-color melting curve analysis method, etc.

[0003] Real-time fluorescence PCR is the most widely used method for nucleic acid detection. The principle of this method is to add an oligonucleic acid probe modified with a fluorescent group and a quenching group at both ends to the PCR reaction system. During the PCR amplification process, the PCR process is detected in real time by monitoring the fluorescence signal. Its operation is simple and it is widely applied. However, this method only detects and analyzes in one dimension, that is, using the fluorescence signal generated by the change in fluorescence. Therefore, only one target nucleic acid can be detected in one fluorescence channel. Therefore, for multiplex nucleic acid detection, due to the limitation of the fluorescence channels, generally only 1 to 6 target nucleic acids can be simultaneously detected in a single tube, and the more the number of fluorescence detection channels, the higher the price of the instrument, increasing the cost of multiplex nucleic acid detection.

[0004] Nucleic acid invasion is a nucleic acid signal amplification detection method. The detection principle of this method is based on the fact that flap endonuclease 1 (FEN1) can recognize the overlapping structure of 3 bases formed by an oligonucleotide and the target DNA, and cut the corresponding oligonucleotide, using the generated Flaps fragment as an identification signal, and then performing signal amplification detection through methods such as secondary cleavage fluorescence resonance energy transfer hairpin probe (FRET probe). Compared with the conventional PCR-based template amplification technology, it has the advantages of high specificity and not being easily cross-contaminated. However, this method has low sensitivity, resulting in fewer applicable scenarios. At the same time, this method also cannot achieve the goal of simultaneously detecting multiple target nucleic acids in a single tube. In addition, although technologies such as high-throughput sequencing method and microarray chip have achieved high-throughput multi-target parallel detection, they are limited in their wide application due to factors such as cumbersome operation, high cost, and long cycle.

[0005] Multi-color melting curve analysis technology combines melting curve analysis on the basis of the real-time fluorescence PCR method, and detects and analyzes in two dimensions, namely fluorescence change and T m value, to achieve multiplex detection in one channel. The principle of common melting curve analysis methods is to use the T generated by the hybridization of a probe with a large number of single-stranded DNAs mThe value is used for melting curve analysis. Therefore, asymmetric PCR is used. By adjusting the concentration ratio of upstream and downstream primers, a large number of single-stranded products complementary to the fluorescent probe are obtained. The asymmetric amplification is combined with the DNA melting temperature to form the characteristic peaks of the melting curve. This method circumvents the fluorescence channel limitation of the PCR instrument in multiplex real-time fluorescence PCR and has the advantages of high throughput, simple operation, low cost, and reliable accuracy. However, the current multi-color melting curve analysis technology still faces numerous challenges. Since asymmetric amplification is linear amplification rather than exponential amplification, it is prone to problems such as low amplification yield and low sensitivity. At the same time, it is difficult to optimize the ratio of upstream and downstream primers, and the design difficulty is relatively large. The molecular beacon used in asymmetric melting curve experiences the processes of hairpin structure, free single strand, hybrid double strand, and free single strand in the reaction. The fluorescence signal consists of two parts: from weak to strong and from strong to weak, and it is prone to problems such as uneven baseline and inverted peaks. Therefore, it is necessary to improve the asymmetric melting curve method in order to improve the detection sensitivity through the symmetric melting curve method.

[0006] Qingge Li et al. (Proceedings of the National Academy of Sciences of the United States of America, 119(9), e2110672119) described a multiplex melting curve analysis method based on mediator probes and fluorescent molecular beacon probes, called MeltArray. This method utilizes the 5'-flap endonuclease activity of Taq DNA polymerase. When the mediator probe binds to the target nucleic acid, the mediator probe can be cleaved to generate a mediator primer that can bind to the fluorescent molecular beacon probe. Subsequently, under the action of Taq DNA polymerase, extension occurs to generate a locally complementary paired fluorescent duplex. Different mediator primers and fluorescent molecular beacon probes extend to generate different fluorescent duplexes. During the melting curve analysis process, different fluorescent duplexes correspond to their respective T m values, and then melting curve peaks are generated at different temperatures, thus realizing the detection of multiple targets.

[0007] However, this method has obvious disadvantages. The design of the fluorescent molecular beacon itself has a strong secondary structure. Although this design makes the 5'-terminal fluorescent group and the 3'-terminal quenching group relatively close, obtaining a low background signal, during the melting curve analysis process of an excessive amount of fluorescent molecular beacon probes, as the temperature increases, the molecular beacon structure is opened, causing the separation of the 5'-terminal fluorescent group and the 3'-terminal quenching group, resulting in an obvious change in the fluorescence signal from weak to strong, manifested as an obvious melting curve peak.

[0008] However, according to the method described by the author, the mediator probe reacts with the target nucleic acid to generate a mediator primer. Different mediator primers are extended with a fluorescent molecular beacon probe to obtain different fluorescent double-stranded bodies. At this time, the distance between the fluorescent group and the quenching group is the farthest, and the fluorescent signal is the strongest. During the melting curve analysis, as the temperature increases, the double-stranded DNA is opened, and the single-stranded DNA is in a coiled state. At this time, the 5'-end fluorescent group and the 3'-end quenching group approach each other, and the fluorescent signal becomes weaker, resulting in a change in the fluorescent signal from strong to weak. It can be found that during the melting curve analysis, the fluorescent molecular beacon probe itself will undergo a change in the fluorescent signal from weak to strong, while the fluorescent double-stranded body obtained by extending the mediator primer with the fluorescent molecular beacon probe will produce a change in the fluorescent signal from strong to weak. These two changes are opposite. In other words, if the melting curve peak generated by the change in the fluorescent signal from strong to weak is defined as a positive peak, then the inverted peak generated by the fluorescent molecular beacon probe itself is inevitable. This will cause the baseline of the target melting curve peak to be uneven, and the fluorescent signals of the inverted peak and the positive peak will interfere with and cancel each other, ultimately affecting the sensitivity and detection rate. Due to the inevitable inverted peak, it will also directly affect the interpretation of the qPCR instrument, resulting in false positives or false negatives, etc.

[0009] Correspondingly, in the methods disclosed in Patent CN108823287B, CN110273012B, CN109988865B, and CN110273013B, the same problem exists when using a fluorescent molecular beacon probe as a detection probe. In addition, in the methods disclosed in the above patents, in addition to using a fluorescent molecular beacon probe as the detection probe, a linear TaqMan probe can also be used. When using a linear TaqMan probe, although problems such as inverted peaks, uneven baselines, and easy misjudgment caused by the secondary structure of the probe itself can be avoided, the excessive mediator probe (uncleaved probe) present in the reaction system will bind to the linear TaqMan probe, thereby changing the distance between the 5'-end fluorescent group and the 3'-end quenching group in the TaqMan probe. During the melting curve analysis, a false positive background peak will be generated.

[0010] In the method disclosed in Patent CN117363767B, the 5'-end fluorescent group and the 3'-end quenching group of the detection probe are modified to a 5'-end quenching group, removing the fluorescent group, and the 5'-end of the mediator probe is modified with a fluorescent group. Although the above changes can correct the problems of inverted peaks and uneven baselines caused by the change in the structure of the detection probe itself, the problem of the excessive mediator probe binding to the detection probe to generate a false positive background peak still cannot be avoided. Summary of the Invention

[0011] To solve the above technical problems, the present invention provides a method for detecting a target nucleic acid to be detected using a melting curve, and the method includes the following steps: Step 1: Design a first primer, a second primer, and a detection probe for the target nucleic acid sequence to be detected, where: a. The first primer sequentially includes a tag sequence, a nicking enzyme specific recognition sequence, and a target nucleic acid recognition sequence from the 5'-end to the 3'-end; the tag sequence cannot bind to the target nucleic acid to be detected, and the nicking enzyme specific recognition sequence refers to the single-stranded oligonucleotide chain sequence in the double-stranded oligonucleotide chain sequence recognized by the nicking enzyme that is not cleaved by the nicking enzyme, and the target nucleic acid recognition sequence refers to the sequence that specifically binds to the target nucleic acid to be detected; b. The second primer, which can specifically bind to the target nucleic acid, and the second primer and the first primer are used to amplify the target nucleic acid to be detected; c. The detection probe, the sequence of which completely includes the continuous sequence composed of the tag sequence and the nicking enzyme specific recognition sequence in the first primer; a reporter group is modified on the detection probe to facilitate the formation of a melting curve peak in the detection stage;

[0012] Step 2: Perform PCR amplification in a PCR amplification system containing the first primer, the second primer, the detection probe, the sample to be detected, the nicking enzyme (Nickingenzymes), and DNA polymerase. If the sample to be detected contains the target nucleic acid to be detected, the target nucleic acid recognition sequence in the first primer specifically binds to the target nucleic acid to be detected and extends to generate a new single strand thereon. The 5'-end of the new single strand is the tag sequence and the nicking enzyme specific recognition sequence of the first primer. The second primer specifically binds to the newly generated single strand and extends to generate a double-stranded amplification product with the 5'-end being the tag sequence and the nicking enzyme specific recognition sequence; the nicking enzyme specifically recognizes the nicking enzyme specific recognition sequence in the double-stranded amplification product and cleaves its complementary sequence to generate a cleavage incision; in the next round of PCR denaturation stage, the cleaved complementary sequence fragment dissociates, and this dissociated fragment becomes a reporter primer; in the annealing stage, DNA polymerase extends and fills in along the incision, and then the nicking enzyme specifically recognizes the nicking enzyme specific recognition sequence in the double-stranded amplification product again and cleaves to generate a new incision and generate a new reporter primer. This cycle repeats, and more reporter primers are generated;

[0013] Step 3: The reporter primer generated in Step 2 is complementary paired with the detection probe to form a double-stranded product, and the melting curve of the double-stranded product is obtained. The melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be detected, thereby realizing the detection of the target nucleic acid to be detected.

[0014] In one embodiment, a method for detecting a target nucleic acid to be detected using a melting curve is provided. The method is used for detecting multiple target nucleic acids to be detected, and the method includes: Step 1: When multiple target nucleic acid sequences A1, A2... An are used as a group of target nucleic acids to be detected simultaneously, corresponding first primers B1, B2... Bn, second primers C1, C2... Cn, and a common detection probe D are designed for each of the target nucleic acid sequences A1, A2... An, where n is an integer not less than 2;

[0015] a. The first primer B1, from the 5'-end to the 3'-end, is successively a tag sequence, a nicking enzyme-specific recognition sequence, and a target nucleic acid recognition sequence; the tag sequence cannot bind to the target nucleic acid to be detected, the nicking enzyme-specific recognition sequence refers to the single-stranded oligonucleotide chain sequence in the double-stranded oligonucleotide chain sequence recognized by the nicking enzyme that is not cleaved by the nicking enzyme, and the target nucleic acid recognition sequence refers to the sequence that specifically binds to the target nucleic acid to be detected; the first primers B1, B2... Bn have the same structure, they contain the same nicking enzyme-specific recognition sequence, the first primers B1, B2... Bn respectively have corresponding target nucleic acid recognition sequences that bind to the target nucleic acid sequences A1, A2... An to be detected, the tag sequences in the first primers B1, B2... Bn are different from each other, and compared with the tag sequence of the first primer B1, the tag sequences of the first primers B2... Bn have at least one SNP site, so as to form melting curve peaks with different T m values;

[0016] b. The second primers C1, C2... Cn can respectively specifically bind to their respective target nucleic acid sequences A1, A2... An to be detected, and the second primers C1, C2... Cn respectively cooperate with the corresponding first primers B1, B2... Bn to amplify the corresponding target nucleic acid sequences A1, A2... An to be detected;

[0017] c. The detection probe D, the sequence of the detection probe D completely contains the continuous sequence composed of the tag sequence and the nicking enzyme-specific recognition sequence in the first primer B1; a reporter group is modified on the detection probe, so as to form a melting curve peak in the detection stage;

[0018] Step 2: Perform PCR amplification in a PCR amplification system containing the first primers B1, B2... Bn, the second primers C1, C2... Cn, a common detection probe D, a test sample, a nicking enzyme, and a DNA polymerase. If the test sample contains multiple of the target nucleic acid sequences A1, A2... An to be detected, the target nucleic acid recognition sequences of B1, B2... Bn in the first primers specifically bind to the corresponding target nucleic acids A1, A2... An to be detected respectively, and new corresponding single strands E1, E2... En are generated by extension on the corresponding target nucleic acids to be detected. The 5'-ends of the single strands E1, E2... En are tag sequences and nicking enzyme specific recognition sequences. The second primers C1, C2... Cn specifically bind to the single strands E1, E2... En generated by the above extension respectively, and double-stranded amplification products F1, F2... Fn with the 5'-ends being the tag sequences and the nicking enzyme specific recognition sequences are generated by extension on them. The nicking enzyme specifically recognizes the nicking enzyme specific recognition sequences in the double-stranded amplification products F1, F2... Fn and cleaves its complementary sequence to produce a cleavage nick. In the denaturation stage of the next round of PCR, the cleaved complementary sequence fragments are released, and these released fragments become the reporter primers G1, G2... Gn. In the annealing stage, the DNA polymerase extends and fills in the gap along the nick, then the nicking enzyme recognizes and cleaves the nicking enzyme specific recognition sequence in the double-stranded amplification product to generate a new nick and new reporter primers G1, G2... Gn. This cycle repeats, and more reporter primers G1, G2... Gn are generated.

[0019] Step 3: The generated reporter primers G1, G2... Gn are respectively complementary paired with the detection probe D to form different double-stranded products H1, H2... Hn. The generated reporter primers G1, G2... Gn are respectively complementary paired with the detection probe D to form different double-stranded products H1, H2... Hn. Melting curves of different double-stranded products H1, H2... Hn are obtained. The melting curves of different double-stranded products H1, H2... Hn are respectively the melting curves of the corresponding target nucleic acids A1, A2... An to be detected, thereby realizing the simultaneous detection of multiple target nucleic acid sequences A1, A2... An to be detected.

[0020] In the above method, when multiple target nucleic acid sequences A1, A2... An are used as a group of target nucleic acids to be detected simultaneously, they are detected in the same fluorescence channel. Of course, in order to further increase the detection throughput, a group of target nucleic acids to be detected simultaneously can be detected separately in multiple fluorescence detection channels. For example, multiple target nucleic acid sequences A1, A2... An are detected in the FAM fluorescence channel, and multiple different target nucleic acid sequences A11, A12... A1n are detected in the VIC fluorescence channel.

[0021] In one embodiment, in step 1, a corresponding Taqman probe is designed for each target nucleic acid sequence to be detected. This Taqman probe is used to generate a real-time amplification curve during the PCR amplification stage to facilitate the detection of the amplification reaction.

[0022] In one embodiment, the detection probe sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

[0023] In one embodiment, the length of the tag sequence part in the first primer is 4 - 30 nt, and the length of the nicking enzyme specific recognition sequence is 1 - 40 nt.

[0024] In one embodiment, the modified reporter group on the detection probe is a pair of quenching group and fluorescent group. Fluorescent groups include various commonly used fluorescent markers currently, but are not limited to these fluorescent markers, such as PacificBlue, Oregon Green, Bodipy FL-X, FAM, VIC, TET, Bodipy R6G-X, JOE, HEX, Cy3, Cy3B, Rhodamine Red, TAMRA, Texas Red-X, ROX, Cy3.5, Cy5, etc.; the quenching groups include various commonly used quenching agents currently, but are not limited to these quenching agents, such as Dabcyl, Eclipse, BHQ-1, BHQ-2, QYS-7, etc.

[0025] In one embodiment, the nicking enzyme includes at least one of Nb.BbvCI, Nb.Bpu10I, Nb.BsmI, Nb.BsrDI, Nb.BssSI, Nb.BtsI, Nb.Mva1269I, Nt.AlwI, Nt.BbvCI, Nt.Bpu10I, Nt.BsmAI, Nt.BspQI, Nt.Bst9I, Nt.BstNBI, Nt.CviPII.

[0026] In one embodiment, the DNA polymerase includes at least one of Taq DNA polymerase, Tth DNA polymerase, TflDNA polymerase, Tfi DNA polymerase, pfu DNA polymerase, KOD DNA polymerase, Tgo DNA, Bst DNA polymerase, BsoBI DNA polymerase, Bacillus stearothermophilus DNA polymerase, or φ29 DNA polymerase.

[0027] In one embodiment, the generated reporter primer G1 is completely complementary to the detection probe D, and the generated reporter primers G2... Gn have different numbers of SNP sites or SNP sites at different positions with respect to the detection probe D, and cannot be completely complementary, thereby forming different double-stranded products H1, H2... Hn with different T m values.

[0028] In one embodiment, there is provided a composition for detecting a target nucleic acid to be measured by melting curve, the composition comprising: a first primer, a second primer and a detection probe designed for the target nucleic acid sequence to be measured, wherein, a. The first primer comprises a tag sequence, a nicking enzyme specific recognition sequence and a target nucleic acid recognition sequence in sequence from the 5'-end to the 3'-end; the tag sequence cannot bind to the target nucleic acid to be measured, and the nicking enzyme specific recognition sequence refers to a single-stranded oligonucleotide sequence in the double-stranded oligonucleotide sequence recognized by the nicking enzyme that is not cleaved by the nicking enzyme, and the target nucleic acid recognition sequence refers to a sequence that specifically binds to the target nucleic acid to be measured;

[0029] b. A second primer, which can specifically bind to the target nucleic acid, and the second primer and the first primer are used to amplify the target nucleic acid to be measured;

[0030] c. A detection probe, the sequence of which completely contains the continuous sequence composed of the tag sequence and the nicking enzyme specific recognition sequence in the first primer; a reporter group is modified on the detection probe to facilitate the formation of a melting curve peak in the detection stage.

[0031] In one embodiment, there is provided a composition for detecting a target nucleic acid to be measured by melting curve, the composition comprising: first primers B1, B2... Bn and second primers C1, C2... Cn and a common detection probe D, where n is an integer not less than 2; when multiple target nucleic acid sequences A1, A2... An to be measured are detected simultaneously as a group, corresponding first primers B1, B2... Bn and second primers C1, C2... Cn and a common detection probe D are designed for each target nucleic acid sequence A1, A2... An respectively;

[0032] a. The first primer B1, from the 5'-end to the 3'-end, successively includes a tag sequence, a nicking enzyme specific recognition sequence, and a target nucleic acid recognition sequence; the tag sequence cannot bind to the target nucleic acid to be detected; the nicking enzyme specific recognition sequence refers to the single-stranded oligonucleotide chain sequence in the double-stranded oligonucleotide chain sequence recognized by the nicking enzyme that is not cleaved by the nicking enzyme; the target nucleic acid recognition sequence refers to the sequence that specifically binds to the target nucleic acid to be detected; the first primers B1, B2... Bn have the same structure, they contain the same nicking enzyme specific recognition sequence, the first primers B1, B2... Bn respectively have corresponding target nucleic acid recognition sequences that bind to the target nucleic acid sequences A1, A2... An to be detected, the tag sequences in the first primers B1, B2... Bn are different from each other, and compared with the tag sequence of the first primer B1, the tag sequences of the first primers B2... Bn have at least one SNP site, so as to form melting curve peaks with different T m values;

[0033] b. The second primers C1, C2... Cn can respectively specifically bind to their respective target nucleic acid sequences A1, A2... An to be detected, and the second primers C1, C2... Cn respectively cooperate with the corresponding first primers B1, B2... Bn to amplify the corresponding target nucleic acid sequences A1, A2... An to be detected;

[0034] c. A detection probe D, the sequence of the detection probe D completely contains the continuous sequence composed of the tag sequence and the nicking enzyme specific recognition sequence in the first primer B1; a reporter group is modified on the detection probe, so as to form a melting curve peak in the detection stage.

[0035] In one embodiment, it further includes Taqman probes corresponding to each target nucleic acid sequence to be detected.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] a. In the technical solution of the present invention, the reporting primer generated in step 2 is complementary to and paired with the detection probe to form a double-stranded product, and a melting curve of the double-stranded product is obtained. The melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be detected, so as to realize the detection of the target nucleic acid to be detected. Therefore, the method of the present invention is a non-target-dependent melting curve method, that is, the sequences of each reporting primer and the detection probe are artificially designed, known or predetermined. Therefore, the melting point (T m value) of each double-stranded body can be calculated in advance. Thus, by detecting the melting point (T mBy using the melting peak of the double-stranded DNA (dsDNA) with a specific melting temperature (Tm value), the presence of the target nucleic acid sequence corresponding to the dsDNA in the sample can be determined. This solves the problem of peak shift in the melting curve caused by the easy mutation of the target nucleic acid sequence to be detected, which is prone to misjudgment. It also solves the problem that the melting curve analysis method is not applicable to RNA samples, greatly increasing the range of samples that can be detected by the melting curve method and significantly improving the detection accuracy. At the same time, since the sequences of the reporter primer and the detection probe are both artificially designed, known, or pre-determined, and the melting temperature of each dsDNA is pre-calculated, the detection throughput can be greatly increased.

[0038] b. In the technical solution of the present invention, the method cleverly utilizes the characteristics that the nicking enzyme recognizes a specific double-stranded oligonucleotide chain sequence (Oligo) but only cuts one of the single-stranded Oligos, and the property that DNA polymerase extends from the 5' end to the 3' end. Specifically, in the technical solution of the present invention, the target nucleic acid recognition sequence in the first primer specifically binds to the target nucleic acid to be detected and generates a new single strand by extension thereon. The 5' end of the new single strand is the tag sequence of the first primer and the specific recognition sequence of the nicking enzyme. The second primer specifically binds to the newly generated single strand by extension and generates a double-stranded amplification product with the 5' end being the tag sequence and the specific recognition sequence of the nicking enzyme. The nicking enzyme specifically recognizes the specific recognition sequence of the nicking enzyme in the double-stranded amplification product and cleaves its complementary sequence to produce a cleavage nick. In the denaturation stage of the next round of PCR, the cleaved complementary sequence fragment dissociates, and this dissociated fragment becomes the reporter primer. In the annealing stage, DNA polymerase extends and fills in the gap along the nick, and then the nicking enzyme specifically recognizes the specific recognition sequence of the nicking enzyme in the double-stranded amplification product again and cleaves to generate a new nick and a new reporter primer. This process repeats in a cycle, generating more reporter primers. Therefore, in the technical solution of the present invention, due to the generation of more reporter primers, a reporter primer cascade amplification system is constructed. Compared with the system of only PCR amplification reaction, this method amplifies the detection signal twice, further improving the detection sensitivity, the detection accuracy rate, and the accuracy.

[0039] c. In the technical solution of the present invention, the target nucleic acid recognition sequence in the first primer specifically binds to the target nucleic acid to be detected, and a new single strand is generated by extension thereon. The second primer specifically binds to the newly generated single strand by extension, and a double-stranded amplification product with the tag sequence and the nicking enzyme specific recognition sequence at the 5' end is generated by extension thereon. The first primer and the second primer are in equal amounts. Therefore, the method for detecting target nucleic acid by melting curve in the present invention is a symmetric amplification melting curve method (an exponential amplification method). Compared with the common asymmetric amplification melting curve method (a linear amplification method), this method utilizes the advantage of exponential amplification of PCR to enrich a large amount of target nucleic acid and improve the detection sensitivity;

[0040] d. In the technical solution of the present invention, the reporting primer generated in step 2 is complementary to the detection probe to form a double-stranded product, and the melting curve of the double-stranded product is obtained. The melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be detected, thereby realizing the detection of the target nucleic acid to be detected. The generation of the reporting primer has a causal relationship with the presence or absence of the target to be detected, that is, the reporting primer will only be generated when the target to be detected exists. Only the reporting primer can bind to the detection probe, and a melting curve peak can be generated in the melting curve analysis stage. Compared with the prior art methods, the common feature of the prior art methods is that the medium probes that bind to the detection probe are all artificially added. However, the medium probes are often in excess, and the excess medium probes will bind to the detection probe to generate false positive background peaks, interfering with the accuracy and correct rate of interpretation. The method of the present invention solves the problem of false positive background peaks well and improves the detection accuracy and correct rate of interpretation;

[0041] e. In the technical solution of the present invention, for the same detection channel, SNP sites with different positions and numbers are ingeniously introduced into the tag sequence part of each tag primer, so that in each detection channel, a single detection probe can realize the detection of multiple target nucleic acids, greatly reducing the number of probes, reducing the detection cost, reducing the complexity of the detection system, and improving the detection accuracy and success rate;

[0042] f. In the technical solution of the present invention, during the melting curve analysis process, the fluorescence signal fluctuations of the reporting primer and the detection probe are reflected in a larger proportion, obtaining a flatter baseline, reducing the appearance of non-specific peaks, and improving the correct rate and accuracy of the detection method. At the same time, this method is not limited by the number of fluorescence channels detected. Different targets are distinguished according to the melting point and fluorescence color, increasing the number of target detections in a single well and improving the detection throughput. The method of the present invention can be used for multiplex target detection. The baselines of the melting peaks can all remain flat, the peaks are higher, and there are no non-specific peaks, with higher sensitivity; at the same time, the detection interval range is also wider, and the melting curve detection T m value range is: 30~90°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings described below are only some embodiments described in the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0044] Figure 1 Schematic diagram of the first primer used in the method of the present invention;

[0045] Figure 2 Schematic diagram of the basic principle of the method of the present invention;

[0046] Figure 3 Schematic diagram of multiple first primers for detecting multiple target nucleic acids to be detected in the method of the present invention;

[0047] Figure 4 Schematic diagram of the basic principle of the method of the present invention for detecting multiple target nucleic acids to be detected;

[0048] Figure 5 Schematic diagram of the detection result of EGFR by the method of the present invention;

[0049] Figure 6 Schematic diagram of the detection result of GAPDH by the method of the present invention;

[0050] Figure 7 Schematic diagram of the detection result of PIK3CA by the method of the present invention;

[0051] Figure 8 Schematic diagram of the triple detection result of EGFR, GAPDH, and PIK3CA by the method of the present invention

[0052] Figure 9 Schematic diagram of the six - fold detection result of EGFR, GAPDH, PIK3CA, BRAF, ACTB, and CDA by the method of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0053] In order to enable those skilled in the art to better understand the technical solutions in the present application, the following will further illustrate the present invention in combination with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts should fall within the protection scope of the present application. In the following embodiments, unless otherwise specified, they are all conventional methods in the art.

[0054] Embodiment 1 Basic Principle of the Present Invention

[0055] As Figure 1 and Figure 2 shown, the present invention provides a method for detecting a target nucleic acid by melting curve, and a method for detecting a single target nucleic acid to be detected by melting curve. The method includes:

[0056] A method for detecting a target nucleic acid to be detected by melting curve, the method including the following steps:

[0057] Step 1: Design a first primer, a second primer and a detection probe for the target nucleic acid sequence to be detected, wherein:

[0058] a. The first primer is, from the 5'-end to the 3'-end, a tag sequence, a nicking enzyme specific recognition sequence and a target nucleic acid recognition sequence in sequence; the tag sequence cannot bind to the target nucleic acid to be detected; the nicking enzyme specific recognition sequence refers to a single-stranded oligonucleotide chain sequence in a double-stranded oligonucleotide chain sequence recognized by the nicking enzyme that is not cleaved by the nicking enzyme; the target nucleic acid recognition sequence refers to a sequence that specifically binds to the target nucleic acid to be detected;

[0059] b. A second primer, the second primer specifically binds to the target nucleic acid to be detected, and the second primer and the first primer are used to amplify the target nucleic acid to be detected;

[0060] c. A detection probe, the sequence of the detection probe includes a continuous sequence composed of the tag sequence and the nicking enzyme specific recognition sequence in the first primer; a reporter group is modified on the detection probe so as to form a melting curve peak in the detection stage;

[0061] Step 2: Perform PCR amplification in a PCR amplification system containing the first primer, the second primer, the detection probe, the sample to be detected, the nicking enzyme and DNA polymerase. If the sample to be detected contains the target nucleic acid to be detected, the target nucleic acid recognition sequence in the first primer specifically binds to the target nucleic acid to be detected and extends on it to generate a new single strand, the 5'-end of which is the tag sequence and the nicking enzyme specific recognition sequence of the first primer. The second primer specifically binds to the new single strand generated by the above extension and extends on it to generate a double-stranded amplification product with the 5'-end being the tag sequence and the nicking enzyme specific recognition sequence; the nicking enzyme specifically recognizes the nicking enzyme specific recognition sequence in the double-stranded amplification product and cleaves its complementary sequence to generate a cleavage incision; in the next round of PCR denaturation stage, the cleaved complementary sequence fragment dissociates, and this dissociated fragment becomes a reporter primer; in the annealing stage, DNA polymerase extends and fills in along the incision, and then the nicking enzyme specifically recognizes the nicking enzyme specific recognition sequence in the double-stranded amplification product again and cleaves to generate a new incision and generate a new reporter primer, and so on in a cycle, and more reporter primers are generated;

[0062] Step 3: The reporting primer generated in Step 2 is complementary paired with the detection probe to form a double-stranded product, and the melting curve of the double-stranded product is obtained. The melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be detected, thereby realizing the detection of the target nucleic acid to be detected.

[0063] Similarly, as Figure 3 and Figure 4 shown, a method for detecting a target nucleic acid to be detected by a melting curve, the method is used for detecting multiple target nucleic acids to be detected, and the method includes:

[0064] Step 1: When multiple target nucleic acid sequences A1, A2... An are target nucleic acids to be detected that are detected simultaneously as a group, corresponding first primers B1, B2... Bn and second primers C1, C2... Cn and a shared detection probe D are designed for each of the target nucleic acid sequences A1, A2... An, where n is an integer not less than 2;

[0065] a. The first primer B1 is, from the 5' end to the 3' end, a tag sequence, a nicking enzyme-specific recognition sequence, and a target nucleic acid recognition sequence in sequence; the tag sequence cannot bind to the target nucleic acid to be detected, and the nicking enzyme-specific recognition sequence refers to a single-stranded oligonucleotide chain sequence in the double-stranded oligonucleotide chain sequence recognized by the nicking enzyme that is not cleaved by the nicking enzyme, and the target nucleic acid recognition sequence refers to a sequence that specifically binds to the target nucleic acid to be detected; the first primers B1, B2... Bn have the same structure, they contain the same nicking enzyme-specific recognition sequence, the first primers B1, B2... Bn respectively have corresponding target nucleic acid recognition sequences that bind to the target nucleic acid sequences A1, A2... An, the tag sequences in the first primers B1, B2... Bn are different from each other, and compared with the tag sequence of the first primer B1, the tag sequences of the first primers B2... Bn have at least one SNP site, so as to facilitate the formation of melting curve peaks with different T m values;

[0066] b. The second primers C1, C2... Cn can respectively specifically bind to their respective target nucleic acid sequences A1, A2... An, and the second primers C1, C2... Cn respectively cooperate with the corresponding first primers B1, B2... Bn to amplify the corresponding target nucleic acid sequences A1, A2... An;

[0067] c. Detection probe D, the sequence of the detection probe D completely contains the continuous sequence composed of the tag sequence and the nicking enzyme-specific recognition sequence in the first primer B1; a reporting group is modified on the detection probe, so as to facilitate the formation of melting curve peaks in the detection stage;

[0068] Step 2: Perform PCR amplification in a PCR amplification system containing the first primers B1, B2... Bn, the second primers C1, C2... Cn, a common detection probe D, a test sample, a nicking enzyme, and a DNA polymerase. If the test sample contains multiple target nucleic acid sequences A1, A2... An among the target nucleic acids to be detected, the target nucleic acid recognition sequences of B1, B2... Bn in the first primers specifically bind to the corresponding target nucleic acids A1, A2... An to be detected respectively, and new corresponding single strands E1, E2... En are extended on the corresponding target nucleic acids to be detected. The 5'-ends of the single strands E1, E2... En are tag sequences and nicking enzyme-specific recognition sequences. The second primers C1, C2... Cn specifically bind to the single strands E1, E2... En extended above respectively, and double-stranded amplification products F1, F2... Fn with the 5'-ends being the tag sequences and the nicking enzyme-specific recognition sequences are extended thereon. The nicking enzyme specifically recognizes the nicking enzyme-specific recognition sequences in the double-stranded amplification products F1, F2... Fn and cleaves their complementary sequences to generate a cleavage nick. In the denaturation stage of the next round of PCR, the cleaved complementary sequence fragments dissociate, and these dissociated fragments become the reporter primers G1, G2... Gn. In the annealing stage, the DNA polymerase extends and fills in the gap along the nick, and then the nicking enzyme recognizes and cleaves the nicking enzyme-specific recognition sequences in the double-stranded amplification products to generate new nicks, generating new reporter primers G1, G2... Gn. This cycle repeats, and more reporter primers G1, G2... Gn are generated;

[0069] Step 3: The generated reporter primers G1, G2... Gn are respectively complementary paired with the detection probe D to form different double-stranded products H1, H2... Hn. Melting curves of different double-stranded products H1, H2... Hn are obtained. The melting curves of different double-stranded products H1, H2... Hn are respectively the melting curves of the corresponding target nucleic acids A1, A2... An to be detected, thereby realizing the simultaneous detection of multiple target nucleic acid sequences A1, A2... An.

[0070] Example 2: EGFR single-target detection experiment

[0071] Taking the detection of EGFR as an example, this example qualitatively detects the EGFR gene in human genomic DNA by the method of the present invention. The specific method includes the following steps:

[0072] I. Primer and probe sequence information

[0073] Design the first primer, the second primer, and the detection probe according to the conserved region of the nucleic acid sequence to be detected. The sequence information is shown in the following table.

[0074] Table 1: Primer, probe sequences and usage concentrations involved in this example

[0075]

[0076] Note: The lowercase letter bases are the tag sequences, the bold lowercase letters are the specific recognition sequences of the nicking enzyme (Nb.BsrDI), and the capital letters are the target nucleic acid recognition sequences.

[0077] II. PCR Amplification Reaction System and Procedure

[0078] The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis. The PCR reaction system includes: 1× PCR buffer, 2 U BioZues® HS Multiplex Taq DNA Polymerase, 1 U Nb.BsrDI, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 100 nM detection probe, and 5 μL human genomic DNA.

[0079] The fluorescence PCR reaction procedure of the present invention is pre-denaturation at 95°C for 1 min; denaturation at 95°C for 5 s, annealing and extension at 60°C for 45 s while collecting fluorescence, repeating 45 cycles; the melting curve procedure is 95°C for 2 min, 30°C for 5 min, and 30 - 90°C for melting curve analysis, detecting the fluorescence signal every 0.04°C. The instrument used in this example is the SLAN 96 Real-Time Fluorescence PCR Instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0080] III. Detection Results

[0081] The EGFR detection result of the method of the present invention is as Figure 5 shown, the melting peak T m value is at 67.1°C. The baseline of this result is flat, the peak value meets the expectation, and there are no non-specific peaks.

[0082] Example 3 GAPDH Single Target Detection Experiment

[0083] In this example, taking the detection of GAPDH as an example, the method of the present invention is used to qualitatively detect the GAPDH gene of human genomic DNA. The specific method includes the following steps:

[0084] I. Primer and Probe Sequence Information

[0085] According to the conserved region of the nucleic acid sequence to be detected, the first primer, the second primer and the detection probe are designed. The sequence information is shown in the following table.

[0086] Table 2: Primer and Probe Sequences and Usage Concentrations Involved in the Example

[0087]

[0088] Note: The lowercase letter bases are the tag sequences, the underlined lowercase letters are the SNP sites of the tags, the bold lowercase letters are the specific recognition sequences of the nicking enzyme (Nb.BsrDI), and the capital letters are the target nucleic acid recognition sequences.

[0089] II. PCR Amplification Reaction System and Procedure

[0090] The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis. The PCR reaction system includes: 1× PCR buffer, 2 U BioZues® HS Multiplex Taq DNA Polymerase, 1 U Nb.BsrDI, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 100 nM detection probe, and 5 μL human genomic DNA.

[0091] The fluorescence PCR reaction procedure of the present invention is pre-denaturation at 95°C for 1 min; denaturation at 95°C for 5 s, annealing and extension at 60°C for 45 s while collecting fluorescence, repeating 45 cycles; the melting curve procedure is 95°C for 2 min, 30°C for 5 min, and 30 - 90°C for melting curve analysis, detecting the fluorescence signal every 0.04°C. The instrument used in this example is the SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0092] III. Detection Results

[0093] The detection result of GAPDH by the method of the present invention is as Figure 6 shown. The melting peak T m value is at 62.4°C. The baseline of this result is flat, the peak value meets the expectation, and there are no non-specific peaks.

[0094] Example 4 PIK3CA Single Target Detection Experiment

[0095] Taking the detection of PIK3CA as an example, this example qualitatively detects the PIK3CA gene of human genomic DNA by the method of the present invention. The specific method includes the following steps:

[0096] I. Primer and Probe Sequence Information

[0097] The first primer, second primer, and detection probe are designed according to the conserved region of the nucleic acid sequence to be detected. The sequence information is shown in the following table.

[0098] Table 3: Primer and Probe Sequences and Usage Concentrations Involved in the Example

[0099]

[0100] Note: The lowercase letter bases are the tag sequences, the underlined lowercase letters are the SNP sites of the tags, the bold lowercase letters are the specific recognition sequences of the nicking enzyme (Nb.BsrDI), and the uppercase letters are the target nucleic acid recognition sequences.

[0101] II. PCR Amplification Reaction System and Procedure

[0102] The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis. The PCR reaction system includes: 1× PCR buffer, 2 U BioZues® HS Multiplex Taq DNA Polymerase, 1 U Nb.BsrDI, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 100 nM detection probe, and 5 μL human genomic DNA.

[0103] The fluorescence PCR reaction procedure of the present invention is pre-denaturation at 95°C for 1 min; denaturation at 95°C for 5 s, annealing and extension at 60°C for 45 s while collecting fluorescence, repeating 45 cycles; the melting curve procedure is 95°C for 2 min, 30°C for 5 min, and 30 - 90°C for melting curve analysis, detecting the fluorescence signal every 0.04°C. The instrument used in this example is the SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0104] III. Detection Results

[0105] The detection result of PIK3CA by the method of the present invention is as Figure 7 shown, the melting peak T m value is at 53.8°C. The baseline of this result is flat, the peak value meets the expectation, and there are no non-specific peaks.

[0106] Example 5 Triple Target Detection Experiment of the Present Invention

[0107] Taking the detection of gDNA as an example, this example qualitatively detects the EGFR, GAPDH, and PIK3CA genes of human genomic DNA by the method of the present invention. The specific method includes the following steps:

[0108] I. Primer and Probe Sequence Information

[0109] Design the first primer, second primer, and detection probe according to the conserved region of the nucleic acid sequence to be detected. The sequence information is shown in the following table.

[0110] Table 4: Primer and Probe Sequences and Usage Concentrations Involved in the Example

[0111]

[0112] Note: The lowercase letter bases are the tag sequences, the underlined lowercase letters are the SNP sites of the tags, the bold lowercase letters are the specific recognition sequences of the nicking enzyme (Nb.BsrDI), and the uppercase letters are the target nucleic acid recognition sequences.

[0113] II. PCR Amplification Reaction System and Procedure

[0114] The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis. The PCR reaction system includes: 1× PCR buffer, 2 U BioZues® HS Multiplex Taq DNA Polymerase, 1 U Nb.BsrDI, 0.2 mM dNTPs, 40 nM of each first primer, 40 nM of each second primer, 300 nM of the detection probe, and 5 μL of human genomic DNA.

[0115] The fluorescence PCR reaction procedure of the present invention is pre-denaturation at 95°C for 1 min; denaturation at 95°C for 5 s, annealing and extension at 60°C for 45 s while collecting fluorescence, repeating 45 cycles; the melting curve procedure is 95°C for 2 min, 30°C for 5 min, and 30 - 90°C for melting curve analysis, detecting the fluorescence signal every 0.04°C. The instrument used in this example is the SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0116] III. Detection Results

[0117] The detection results of genomic DNA EGFR, GAPDH, and PIK3CA are as Figure 8 shown. The melting peak T m values are respectively: 67.9°C, 62.3°C, 54.8°C. The baseline of this result is flat, 3 targets can be detected simultaneously, the peak values meet expectations, and there are no non-specific peaks.

[0118] Example VI Multiplex Target Detection Experiment of the Present Invention

[0119] Taking the detection of gDNA as an example, this example qualitatively detects the human genomic DNA EGFR, GAPDH, PIK3CA, BRAF, ACTB, and CDA genes by the method of the present invention. The specific method includes the following steps:

[0120] I. Primer and Probe Sequence Information

[0121] The first primer, the second primer, and the detection probe are designed according to the conserved regions of the nucleic acid sequence to be detected. The sequence information is shown in the following table.

[0122] Table 5: Primer and Probe Sequences and Usage Concentrations Involved in the Example

[0123]

[0124] Note: The lowercase letter bases are the tag sequences, the underlined lowercase letters are the SNP sites of the tags, the bold lowercase letters are the specific recognition sequences of the nicking enzyme (Nb.BsrDI), and the uppercase letters are the target nucleic acid recognition sequences.

[0125] II. PCR Amplification Reaction System and Procedure

[0126] The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis. The PCR reaction system includes: 1× PCR buffer, 2 U BioZues® HS Multiplex Taq DNA Polymerase, 1 U Nb.BsrDI, 0.2 mM dNTPs, 40 nM of each first primer, 40 nM of each second primer, 300 nM of each of the two detection probes, and 5 μL of human genomic DNA.

[0127] The fluorescence PCR reaction procedure of the present invention is pre-denaturation at 95°C for 1 min; denaturation at 95°C for 5 s, annealing and extension at 60°C for 45 s while collecting fluorescence, repeating 45 cycles; the melting curve procedure is 95°C for 2 min, 30°C for 5 min, and 30 - 90°C for melting curve analysis, detecting the fluorescence signal every 0.04°C. The instrument used in this example is the SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0128] III. Detection Results

[0129] The detection results of genomic DNA for EGFR, GAPDH, PIK3CA, BRAF, ACTB, and CDA are as Figure 6 shown. The melting peak T m values in the Cy5 channel are respectively: 68.2°C, 62.6°C, 55.1°C, and the melting peak T m values in the ROX channel are respectively: 73.8°C, 65.9°C, 55.5°C. The baseline of this result is flat, 6 targets can be detected simultaneously, the peak values meet the expectations, and there are no non-specific peaks.

[0130] It should be understood that the present invention disclosed is not limited to the specific methods, protocols, and substances described, as these can vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is only limited by the appended claims.

[0131] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also encompassed by the appended claims.

Claims

1. A method for detecting a target nucleic acid using a melting curve, characterized in that: The method comprises the following steps: Step 1: Designing a first primer, a second primer and a detection probe for the target nucleic acid sequence to be detected, wherein: a. The first primer comprises a tag sequence, a nickase-specific recognition sequence and a target nucleic acid recognition sequence from the 5' end to the 3' end; the tag sequence cannot bind to the target nucleic acid to be detected, the nickase-specific recognition sequence refers to a single-stranded oligonucleotide chain sequence that is not cut by the nickase in the double-stranded oligonucleotide chain sequence recognized by the nickase, and the target nucleic acid recognition sequence refers to a sequence that specifically binds to the target nucleic acid to be detected; b. a second primer, wherein the second primer specifically binds to the target nucleic acid to be detected, and the second primer and the first primer are used to amplify the target nucleic acid to be detected; c. A detection probe, wherein the sequence of the detection probe comprises a continuous sequence consisting of a tag sequence in the first primer and a nicking enzyme-specific recognition sequence; the detection probe is modified with a reporter group to form a melting curve peak in the detection stage; the reporter group modified on the detection probe is a set of paired quenching groups and fluorescent groups; Step 2: Perform PCR amplification in a PCR amplification system containing the first primer, the second primer and the detection probe, the sample to be tested, the nickase and the DNA polymerase. If the sample to be tested contains the target nucleic acid to be tested, the target nucleic acid recognition sequence in the first primer specifically binds to the target nucleic acid to be tested and extends thereon to generate a new single strand, the 5' end of the new single strand is the tag sequence and the nickase specific recognition sequence of the first primer, the second primer specifically binds to the new single strand generated by the extension, and extends thereon to generate a 5' end of the tag sequence and the nickase specific recognition sequence. The double-stranded amplification product with a specific sequence is prepared; the nickase specifically recognizes the nickase specific recognition sequence in the double-stranded amplification product, and performs enzyme cleavage on its complementary sequence to generate an enzyme cleavage incision; in the next round of PCR denaturation phase, the cut complementary sequence fragment is freed, and the free fragment becomes a reporter primer; in the annealing phase, the DNA polymerase extends and fills along the incision, and then the nickase specifically recognizes the nickase specific recognition sequence in the double-stranded amplification product again and cuts it, generating a new incision, generating a new reporter primer, and repeating the cycle to generate more reporter primers; Step 3: The reporter primer generated in step 2 is complementary paired with the detection probe to directly form a double-stranded product, and a melting curve of the double-stranded product is obtained. The melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be detected, thereby realizing the detection of the target nucleic acid to be detected.

2. A method for detecting a target nucleic acid using a melting curve, characterized in that: The method is used for detecting a plurality of target nucleic acids to be detected, and the method comprises: Step 1: When a plurality of target nucleic acid sequences A1, A2...An are used as a group of target nucleic acids to be detected simultaneously, a corresponding first primer B1, B2...Bn and a second primer C1, C2...Cn and a common detection probe D are designed for each target nucleic acid sequence A1, A2...An, wherein n is an integer not less than 2; a. The first primer B1 has a tag sequence, a nickase-specific recognition sequence and a target nucleic acid recognition sequence from the 5' end to the 3' end; the tag sequence cannot bind to the target nucleic acid to be detected, the nickase-specific recognition sequence refers to a single-stranded oligonucleotide chain sequence that is not cut by the nickase in the double-stranded oligonucleotide chain sequence recognized by the nickase, and the target nucleic acid recognition sequence refers to a sequence that specifically binds to the target nucleic acid to be detected; the first primers B1, B2...Bn have the same structure, they contain the same nickase-specific recognition sequence, the first primers B1, B2...Bn have corresponding target nucleic acid recognition sequences that bind to the target nucleic acid sequences A1, A2...An to be detected, respectively, the tag sequences in the first primers B1, B2...Bn are different, and the tag sequences of the first primers B2...Bn have at least one SNP site compared to the tag sequence of the first primer B1, so as to form a sequence with different T m melting curve peaks with values ​​of b. The second primers C1, C2 ... Cn are capable of specifically binding to the respective target nucleic acid sequences A1, A2 ... An to be detected, and the second primers C1, C2 ... Cn are respectively combined with the corresponding first primers B1, B2 ... Bn to amplify the corresponding target nucleic acid sequences A1, A2 ... An to be detected; c. a detection probe D, wherein the sequence of the detection probe D completely comprises a continuous sequence consisting of the tag sequence and the nicking enzyme specific recognition sequence in the first primer B1; the detection probe D is modified with a reporter group to form a melting curve peak in the detection stage; the reporter group modified on the detection probe D is a pair of quenching groups and fluorescent groups; Step 2: PCR amplification is performed in a PCR amplification system containing the first primers B1, B2...Bn, the second primers C1, C2...Cn and a common detection probe D, a sample to be tested, a nickase and a DNA polymerase. If the sample to be tested contains multiple target nucleic acid sequences A1, A2...An, the target nucleic acid recognition sequences of the first primers B1, B2...Bn specifically bind to the corresponding target nucleic acids A1, A2...An, and extend them on the corresponding target nucleic acids to be tested to generate new corresponding single-stranded E1, E2...En, the 5' end of the single-stranded E1, E2...En is a tag sequence and a nickase-specific recognition sequence, the second primers C1, C2...Cn specifically bind to the single-stranded E1, E2...En generated by the extension, and extend them thereon to generate double-stranded amplification products F1, F2...Fn whose 5' end is the tag sequence and the nickase-specific recognition sequence; the nickase specifically recognizes the double-stranded amplification product F1, The nickase specifically recognizes the sequence in F2...Fn and digests its complementary sequence to generate an enzyme digestion incision; in the next round of PCR denaturation stage, the cut complementary sequence fragment is freed, and the freed fragment becomes the reporter primer G1, G2...Gn; in the annealing stage, the DNA polymerase extends and fills along the incision, and then the nickase specifically recognizes the sequence in the double-stranded amplification product and digests it to generate a new incision, thereby generating new reporter primers G1, G2...Gn, and the cycle is repeated to generate more reporter primers G1, G2...Gn; Step 3: The generated reporter primers G1, G2...Gn are complementary paired with the detection probe D respectively to directly form different double-stranded products H1, H2...Hn, and different melting curves of the double-stranded products H1, H2...Hn are obtained. The melting curves of the different double-stranded products H1, H2...Hn are respectively the melting curves of the corresponding target nucleic acids A1, A2...An to be detected, thereby realizing the simultaneous detection of multiple target nucleic acid sequences A1, A2...An to be detected.

3. The method for detecting a target nucleic acid using a melting curve according to claim 1 or 2, characterized in that: In step 1, a corresponding Taqman probe is designed for each target nucleic acid sequence to be detected.

4. The method for detecting a target nucleic acid using a melting curve according to claim 1 or 2, characterized in that: The detection probe sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

5. The method for detecting a target nucleic acid using a melting curve according to claim 1 or 2, characterized in that: The length of the tag sequence portion in the first primer is 4 to 30 nt, and the length of the nicking enzyme specific recognition sequence is 1 to 40 nt.

6. The method for detecting a target nucleic acid using a melting curve according to claim 1 or 2, characterized in that: The nicking enzyme includes at least one of Nb.BbvCI, Nb.Bpu10I, Nb.BsmI, Nb.BsrDI, Nb.BssSI, Nb.BtsI, Nb.Mva1269I, Nt.AlwI, Nt.BbvCI, Nt.Bpu10I, Nt.BsmAI, Nt.BspQI, Nt.Bst9I, Nt.BstNBI, and Nt.CviPII.

7. The method for detecting a target nucleic acid using a melting curve according to claim 1 or 2, characterized in that: The DNA polymerase includes at least one of Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tfi DNA polymerase, pfu DNA polymerase, KOD DNA polymerase, Tgo DNA, Bst DNA polymerase, BsoBI DNA polymerase, Bacillus stearothermophilus DNA polymerase or φ29 DNA polymerase.

8. The method for detecting a target nucleic acid using a melting curve according to claim 2, wherein: The generated reporter primer G1 is completely complementary to the detection probe D, and the generated reporter primers G2...Gn have different numbers of SNP sites or SNP sites at different positions with the detection probe D, and cannot be completely complementary to each other, thereby forming different double-stranded products H1, H2...Hn with different T m value.

9. A kit for detecting a target nucleic acid using a melting curve, characterized in that: The kit comprises: a first primer, a second primer and a detection probe designed for the target nucleic acid sequence to be detected, wherein: a. the first primer comprises a tag sequence, a nickase-specific recognition sequence and a target nucleic acid recognition sequence from the 5' end to the 3' end; the tag sequence cannot bind to the target nucleic acid to be detected, the nickase-specific recognition sequence refers to a single-stranded oligonucleotide chain sequence that is not cut by the nickase in a double-stranded oligonucleotide chain sequence recognized by the nickase, and the target nucleic acid recognition sequence refers to a sequence that specifically binds to the target nucleic acid to be detected; b. a second primer capable of specifically binding to the target nucleic acid, wherein the second primer and the first primer are used to amplify the target nucleic acid to be detected; c. A detection probe, wherein the sequence of the detection probe completely includes a continuous sequence consisting of the label sequence in the first primer and the nicking enzyme specific recognition sequence; the detection probe is modified with a reporter group to facilitate the formation of a melting curve peak during the detection stage; the reporter group modified on the detection probe is a set of paired quenching groups and fluorescent groups.

10. A kit for detecting a target nucleic acid using a melting curve, characterized in that: The kit comprises: first primers B1, B2...Bn and second primers C1, C2...Cn and a common detection probe D, wherein n is an integer not less than 2; when a plurality of target nucleic acid sequences A1, A2...An are used as a group of target nucleic acids to be detected simultaneously, a corresponding first primer B1, B2...Bn and a second primer C1, C2...Cn and a common detection probe D are designed for each target nucleic acid sequence A1, A2...An; a. The first primer B1 has a tag sequence, a nickase-specific recognition sequence and a target nucleic acid recognition sequence from the 5' end to the 3' end; the tag sequence cannot bind to the target nucleic acid to be detected, the nickase-specific recognition sequence refers to a single-stranded oligonucleotide chain sequence that is not cut by the nickase in the double-stranded oligonucleotide chain sequence recognized by the nickase, and the target nucleic acid recognition sequence refers to a sequence that specifically binds to the target nucleic acid to be detected; the first primers B1, B2...Bn have the same structure, they contain the same nickase-specific recognition sequence, the first primers B1, B2...Bn have corresponding target nucleic acid recognition sequences that bind to the target nucleic acid sequences A1, A2...An to be detected, respectively, the tag sequences in the first primers B1, B2...Bn are different, and the tag sequences of the first primers B2...Bn have at least one SNP site compared to the tag sequence of the first primer B1, so as to form a sequence with different T m melting curve peaks with values ​​of b. The second primers C1, C2 ... Cn are capable of specifically binding to the respective target nucleic acid sequences A1, A2 ... An to be detected, and the second primers C1, C2 ... Cn are respectively combined with the corresponding first primers B1, B2 ... Bn to amplify the corresponding target nucleic acid sequences A1, A2 ... An to be detected; c. A detection probe D, wherein the sequence of the detection probe D completely comprises a continuous sequence consisting of the label sequence and the nicking enzyme-specific recognition sequence in the first primer B1; the detection probe is modified with a reporter group to facilitate the formation of a melting curve peak during the detection stage; the reporter group modified on the detection probe D is a set of paired quenching groups and fluorescent groups.

11. The kit according to claim 9 or 10, characterized in that Also included is a Taqman probe corresponding to each target nucleic acid sequence to be detected.

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