Method for detecting target nucleic acid to be detected through melting curve and kit thereof

Abstract

The invention provides a method for detecting to-be-detected target nucleic acid through a melting curve and a kit thereof, and the method comprises the following steps: designing a first primer, a second primer and a detection probe according to a to-be-detected target nucleic acid sequence, pCR amplification is performed in a PCR amplification system containing the first primer, the second primer, the detection probe, a to-be-detected sample, restriction endonuclease and DNA polymerase, a generated report primer and the detection probe are complementarily paired to form a double-chain product, a melting curve of the double-chain product is obtained, and the melting curve of the double-chain product is calculated. The melting curve of the double-chain product is a melting curve corresponding to the target nucleic acid to be detected. The method is a non-target-dependent melting curve method, the melting point (Tm value) of each duplex can be calculated in advance, and the problems of melting curve peak offset and easy misjudgment caused by easy mutation of a target nucleic acid sequence to be detected are solved.

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 through a melting curve and a kit thereof. Background Art

[0002] Nucleic acid detection plays an increasingly important role in molecular diagnosis, molecular biology research, animal and plant quarantine, and food safety testing. Currently, common nucleic acid detection methods mainly include real-time fluorescence PCR, high-throughput sequencing, microarray chips, nucleic acid invasion detection methods, and multicolor melting curve analysis.

[0003] Real-time fluorescence PCR is the most widely used method for nucleic acid detection. The principle of this method is to add oligonucleic acid probes modified with fluorescent groups and quenching groups at both ends to the PCR reaction system. During the PCR amplification process, the PCR process is detected in real time by monitoring the fluorescent signal. It is easy to operate and widely used. However, this method is only in one dimension, that is, the fluorescent signal generated by the fluorescence change is used for detection and analysis. Therefore, one fluorescent channel can only detect one target nucleic acid. Therefore, for multiple nucleic acid detection, due to the limitation of the fluorescent channel, it is generally only possible to detect 1 to 6 target nucleic acids in a single tube at the same time. The more fluorescent detection channels there are, the higher the price of the instrument, which increases the cost of multiple 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 endonucleases 1 (FEN1) can recognize the overlapping structure of three bases between oligonucleotides and target DNA, cut the corresponding oligonucleotides, use the generated Flaps fragments as recognition signals, and then perform signal amplification detection through secondary cleavage fluorescence resonance transfer hairpin probes (FRET probes) and other methods. Compared with conventional PCR-based template amplification technology, it has the advantages of high specificity and low cross-contamination. However, this method has low sensitivity, resulting in fewer application scenarios. At the same time, this method cannot achieve the goal of detecting multiple target nucleic acids in a single tube at the same time. In addition, although high-throughput sequencing methods, microarray chips and other technologies have achieved high-throughput multi-target parallel detection, their widespread application is limited by factors such as cumbersome operation, high cost and long cycle.

[0005] Multicolor melting curve analysis technology combines melting curve analysis with real-time fluorescence PCR method to analyze the fluorescence changes and T m The principle of the common melting curve analysis method is to use the T value generated by the hybridization of the probe with a large number of DNA single strands. mThe melting curve analysis is performed based on the value. Therefore, asymmetric PCR is used to obtain a large number of single-stranded products complementary to the fluorescent probe by adjusting the ratio of upstream and downstream primer concentrations, and asymmetric amplification is combined with the DNA melting temperature to form a characteristic peak of the melting curve. This method avoids the limitation of the fluorescence channel of the PCR instrument during real-time fluorescence PCR multiple detection, and has the advantages of large flux, simple operation, low cost, and reliable accuracy. However, the current multicolor melting curve analysis technology is still facing many challenges. Since asymmetric amplification belongs to linear amplification, non-exponential amplification is prone to low amplification yield and low sensitivity. At the same time, the ratio of upstream and downstream primers is difficult to optimize, and the design is difficult. The molecular beacon used in asymmetric melting curve undergoes a process of hairpin structure, free single strand, hybrid double strand, and free single strand in the reaction. The fluorescence signal is from weak to strong and from strong to weak, and it is easy to have problems such as uneven baseline and inverted peak. Therefore, it is necessary to improve the asymmetric melting curve method in order to improve the detection sensitivity by the method of symmetrical melting curve.

[0006] Qingge Li et al. (Proceedings of the National Academy of Sciences of the United States of America, 119(9), e2110672119) described a multiple melting curve analysis method based on mediator probes and fluorescent molecular beacon probes, called MeltArray. This method utilizes the 5' end flap endonuclease activity of Taq DNA polymerase. When the mediator probe binds to the target nucleic acid, it can cut the mediator probe to produce a mediator primer that can bind to the fluorescent molecular beacon probe. Subsequently, under the action of Taq DNA polymerase, it is extended to produce a locally complementary fluorescent duplex. Different mediator primers and fluorescent molecular beacon probes are extended to produce different fluorescent duplexes. During the melting curve analysis process, different fluorescent duplexes correspond to their respective T m values, thereby generating melting curve peaks at different temperatures, thereby achieving multi-target detection.

[0007] However, this method has obvious disadvantages. The design of the fluorescent molecular beacon itself has a strong secondary structure. Although the design makes the 5' end fluorescent group and the 3' end quenching group close to each other, thus obtaining a lower background signal, the excessive fluorescent molecular beacon probe will open up the molecular beacon structure as the temperature rises during the melting curve analysis, causing the 5' end fluorescent group and the 3' end quenching group to separate, resulting in an obvious change in the fluorescence signal from weak to strong, which is 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 produce a mediator primer, and different mediator primers extend with the fluorescent molecular beacon probe to obtain different fluorescent duplexes. 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 rises, the double-stranded DNA is opened, and the single-stranded DNA is in a curled state. At this time, the 5'-end fluorescent group and the 3'-end quenching group are close to each other, and the fluorescent signal weakens, resulting in a change from strong to weak fluorescent signal. It can be found that during the melting curve analysis, the fluorescent molecular beacon probe itself will undergo a change in fluorescent signal from weak to strong, while the fluorescent duplex obtained by extending the mediator primer and the fluorescent molecular beacon probe will produce a change in fluorescent signal from strong to weak, and the two changes are opposite. In other words, if the melting curve peak generated by the change in 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 each other and cancel each other, ultimately affecting the sensitivity and detection rate. The inevitable inverted peaks will directly affect the interpretation of the qPCR instrument, resulting in misjudgment or missed judgment.

[0009] Correspondingly, the methods disclosed in patents CN108823287B, CN110273012B, CN109988865B and CN110273013B all have the same problem when using fluorescent molecular beacon probes as detection probes. In addition, in the methods disclosed in the above patents, in addition to using fluorescent molecular beacon probes, linear TaqMan probes can also be used as detection probes. When using linear TaqMan probes, although the problems of inverted peaks, uneven baselines and easy misjudgment caused by the secondary structure of the probe itself can be avoided, the excess intermediate probes (probes that have not been cut) in the reaction system will combine with the linear TaqMan probes, thereby changing the distance between the 5' end fluorescent group and the 3' end quenching group in the TaqMan probe, and a false positive background peak will be generated during the melting curve analysis.

[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 the 5'-end quenching group, the fluorescent group is removed, 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 changes in the structure of the detection probe itself, they still cannot avoid the problem of false positive background peaks caused by excessive mediator probes combining with the detection probe. Summary of the invention

[0011] In order to solve the above technical problems, the present invention provides a method for detecting a target nucleic acid to be detected by a melting curve, the method comprising 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 is composed of a label sequence, a restriction endonuclease-specific recognition sequence and a target nucleic acid recognition sequence from the 5' end to the 3' end; the label sequence cannot bind to the target nucleic acid to be detected, the restriction endonuclease-specific recognition sequence refers to any single-stranded oligonucleotide chain sequence in a double-stranded oligonucleotide chain sequence recognized by a restriction endonuclease, 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, the second primer 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. a detection probe, the sequence of the detection probe completely includes a continuous sequence composed of the label sequence and the restriction endonuclease-specific recognition sequence in the first primer; the detection probe is modified with a reporter group to form a melting curve peak in the detection stage; Step 2: PCR amplification is performed in a PCR amplification system containing the first primer, the second primer and the detection probe, the sample to be tested, a restriction endonuclease and a 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 restriction endonuclease 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 double-stranded amplification product whose 5' end is the tag sequence and the restriction endonuclease specific recognition sequence; the restriction endonuclease will specifically recognize the restriction endonuclease specific recognition sequence in the double-stranded amplification product, and perform enzyme cleavage, respectively generating an enzyme cleavage incision in each double-stranded oligonucleotide chain; in the next round of PCR denaturation stage, the cut double-stranded oligonucleotide chain fragments are freed, and the fragments in the free fragments that can be complementary to the detection probe become reporter primers; the reporter primer can extend along the detection probe to form a new double-stranded oligonucleotide chain; Step 3: The reporter primer generated in step 2 is complementary paired and extended 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, thereby realizing the detection of the target nucleic acid to be detected.

[0012] In one embodiment, a method for detecting a target nucleic acid to be detected by melting curve is provided, the method being used for detecting a plurality of target nucleic acids to be detected, the method comprising: 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 comprises a tag sequence, a restriction endonuclease 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 restriction endonuclease specific recognition sequence refers to any single-stranded oligonucleotide chain sequence in the double-stranded oligonucleotide chain sequence recognized by the restriction endonuclease, 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 restriction endonuclease 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, 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 in the first primer B1 and a restriction endonuclease-specific recognition sequence; the detection probe is modified with a reporter group to form a melting curve peak in the detection stage; 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 restriction endonuclease and a DNA polymerase. If the sample to be tested contains multiple target nucleic acid sequences A1, A2...An to be tested, the target nucleic acid recognition sequences of the first primers B1, B2...Bn specifically bind to the corresponding target nucleic acids A1, A2...An to be tested, and extend 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 restriction endonuclease specific recognition sequence, the second primers C1, C2...Cn specifically bind to the single-stranded E1, E2...En generated by the extension, and extend thereon to generate double-stranded amplification products F1, F2...Fn whose 5' end is the tag sequence and the restriction endonuclease specific recognition sequence; the restriction endonuclease will specifically recognize the double-stranded amplification product F1, The restriction endonucleases in F2...Fn specifically recognize the sequences and perform enzyme cleavage, respectively generating an enzyme cleavage incision in the double-stranded oligonucleotide chain; in the next round of PCR denaturation stage, the cut double-stranded oligonucleotide chain fragments are freed, and the fragments in the free fragments that can be complementary to the detection probe become reporter primers G1, G2...Gn; the reporter primers can extend along the detection probe to form new double-stranded oligonucleotide chains; Step 3: The generated reporter primers G1, G2...Gn are respectively complementary paired with the detection probe D and extended 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, 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.

[0013] 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 in multiple fluorescence detection fluorescence channels respectively. 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.

[0014] In one embodiment, in step 1, a corresponding Taqman probe is designed for each target nucleic acid sequence to be detected. The Taqman probe is used to generate a real-time amplification curve during the PCR amplification stage to facilitate detection of the progress of the amplification reaction.

[0015] In one embodiment, the detection probe sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

[0016] In one embodiment, the length of the tag sequence portion in the first primer is 4 to 30 nt, and the length of the restriction endonuclease specific recognition sequence is 1 to 40 nt.

[0017] In one embodiment, the reporter group modified on the detection probe is a pair of quencher groups and fluorescent groups. The fluorescent groups include various fluorescent markers commonly used at present, 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 quencher groups include various quenchers commonly used at present, but are not limited to these quenchers, such as Dabcyl, Eclipse, BHQ-1, BHQ-2, QYS-7, etc.

[0018] In one embodiment, the restriction endonuclease comprises at least one of the following restriction endonucleases: ApeKI (no inactivation buffer special), ApoI (80°C for 20 minutes buffer special), BclI (no inactivation buffer special), BfuAI (60°C for 20 minutes buffer special), BsaBI (80°C for 20 minutes), BsaJI (80°C for 20 minutes), BsaWI (80°C for 20 minutes), BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BsmBI-v2, BsmFI, BsmI, BspQI, BsrI, BssHII, BstAPI, BstBI, BstEII, BstNI, BstUI, BstYI, BtgZI, BtsCI, Bt sIMutI, FatI, FauI, MwoI, PI-PspI, PspGI, SfiI, SmlI, TaqI-v2, TfiI, TseI, Tsp45I, TspMI, TspRI, Tth111I, EcoRI, EcoRV, HindIII, KpnI, XbaI, XhoI, XmaI.

[0019] In one embodiment, the DNA polymerase comprises at least one of the following DNA polymerases: 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.

[0020] In one embodiment, the generated reporter primer G1 is fully complementary to the detection probe D and is extended, 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 fully complementary to each other, thereby forming different double-stranded products H1, H2...Hn with different T m value.

[0021] In one embodiment, a composition for detecting a target nucleic acid to be detected by a melting curve is provided, the composition comprising: 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 restriction endonuclease 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 restriction endonuclease specific recognition sequence refers to any single-stranded oligonucleotide chain sequence in a double-stranded oligonucleotide chain sequence recognized by a restriction endonuclease, 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, the sequence of which completely comprises a continuous sequence consisting of the tag sequence in the first primer and a restriction endonuclease-specific recognition sequence; the detection probe is modified with a reporter group to form a melting curve peak during the detection stage.

[0022] In one embodiment, a composition for detecting a target nucleic acid to be detected by a melting curve is provided, the composition comprising: a first primer B1, B2 ... Bn and a second primer 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 comprises a tag sequence, a restriction endonuclease 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 restriction endonuclease specific recognition sequence refers to any single-stranded oligonucleotide chain sequence in the double-stranded oligonucleotide chain sequence recognized by the restriction endonuclease, 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 restriction endonuclease 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, 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. Detection probe D, the sequence of which completely includes a continuous sequence consisting of the tag sequence in the first primer B1 and the restriction endonuclease specific recognition sequence; the detection probe is modified with a reporter group to form a melting curve peak in the detection stage.

[0023] In one embodiment, it also includes a Taqman probe corresponding to each target nucleic acid sequence to be detected.

[0024] Compared with the prior art, the present invention has the following advantages: a. In the technical solution of the present invention, the reporter primer generated in step 2 is complementary to the detection probe and extended 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, thereby realizing 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 sequence of each reporter primer and detection probe is artificially designed, known or predetermined, and therefore, the melting point (T) of each duplex can be calculated in advance. m Thus, by detecting the melting point (T m The presence of the target nucleic acid sequence corresponding to the duplex in the sample can be determined by looking at the melting peak with a value of 0.05. This solves the problem of melting curve peak shift and misjudgment caused by the easy mutation of the target nucleic acid sequence to be tested, and well solves the problem that the melting curve analysis method is not suitable for RNA samples, greatly increases the range of samples detected by the melting curve method, and greatly improves the accuracy of detection; at the same time, since the sequences of the reporter primers and the detection probes are artificially designed, known or predetermined, the melting points of each duplex are calculated in advance, so the detection throughput can be greatly increased; b. In the technical scheme 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 extends thereon to generate a new single strand, and the second primer specifically binds to the new single strand generated by the extension and extends thereon to generate a double-stranded amplification product whose 5' end is the tag sequence and the restriction endonuclease specific recognition sequence; the first primer and the second primer are equal in amount, so the method of detecting target nucleic acid by melting curve of the present invention is a melting curve method of symmetric amplification (an exponential amplification method), compared with the common melting curve method of asymmetric amplification (a linear amplification method), this method utilizes the advantage of PCR exponential amplification, can enrich a large amount of target nucleic acid, and improve the sensitivity of detection; c. In the technical solution of the present invention, the reporter primer generated in step 2 is complementary paired and extended 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, thereby realizing the detection of the target nucleic acid to be detected. The generation of the reporter primer is causally related to the presence or absence of the target to be detected, that is, the reporter primer will be generated only when the target to be detected exists, and only when the reporter primer can bind to the detection probe can a melting curve peak 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 media probes combined with the detection probes are artificially added, but the media probes are often excessive, and the excessive media probes will combine with the detection probes to produce false positive background peaks, interfering with the accuracy and accuracy of the judgment, while the method of the present invention solves the problem of false positive background peaks well, and improves the accuracy of detection and the accuracy of judgment; d. In the technical solution of the present invention, for the same detection channel, SNP sites of different positions and numbers are cleverly introduced into the tag sequence part of each tag primer, so that in each detection channel, a single detection probe can detect multiple target nucleic acids, which greatly reduces the number of probes, reduces the detection cost, reduces the complexity of the detection system, and improves the detection accuracy and success rate; e. In the technical solution of the present invention, a greater proportion of the fluorescence signal fluctuations of the reporter primer and the detection probe are reflected during the melting curve analysis process, a flatter baseline is obtained, the appearance of non-specific peaks is reduced, and the correctness and accuracy of the detection method are improved. At the same time, this method is not limited to the number of fluorescent channels detected. Different targets are distinguished according to melting point and fluorescent color, the number of targets detected in a single well is increased, and the detection throughput is improved. The method of the present invention can be used for multiple target detection, the baseline of the melting peak can be kept flat, the peak value is higher, there is no non-specific peak, and it has higher sensitivity; at the same time, the detection range is wider, and the melting curve detection T m The value range is: 30~90℃. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 is a schematic diagram of the first primer used in the method of the present invention; Figure 2 It is a schematic diagram of the basic principle of the method of the present invention; Figure 3 A schematic diagram of multiple first primers for detecting multiple target nucleic acids to be detected by the method of the present invention; Figure 4 A schematic diagram of the basic principle of the method of the present invention for detecting multiple target nucleic acids to be detected; Figure 5 This is a schematic diagram of the detection results of EGFR by the method of the present invention; Figure 6 This is a schematic diagram of the detection results of GAPDH by the method of the present invention; Figure 7 This is a schematic diagram of the detection results of PIK3CA by the method of the present invention; Figure 8 Schematic diagram of the triple detection results of EGFR, GAPDH and PIK3CA by the method of the present invention Fig. 9 This is a schematic diagram of the results of the six-fold detection of EGFR, GAPDH, PIK3CA, BRAF, ACTB, and CDA by the method of the present invention. DETAILED DESCRIPTION

[0027] In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described below in conjunction 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 making creative work should belong to the scope of protection of the present application. In the following embodiments, unless otherwise specified, they are all conventional methods in the art.

[0028] Embodiment 1 Basic principle of the present invention like Figure 1 and Figure 2 As shown, the present invention provides a method for detecting a target nucleic acid using a melting curve, and a method for detecting a single target nucleic acid to be detected using a melting curve, the method comprising: A method for detecting a target nucleic acid to be detected by melting curve, the method comprising 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 restriction endonuclease 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 restriction endonuclease specific recognition sequence refers to any single-stranded oligonucleotide chain sequence in the double-stranded oligonucleotide chain sequence recognized by the restriction endonuclease, 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 the tag sequence in the first primer and a restriction endonuclease-specific recognition sequence; the detection probe is modified with a reporter group to facilitate the formation of a melting curve peak in the detection stage; Step 2: PCR amplification is performed in a PCR amplification system containing the first primer, the second primer and the detection probe, the sample to be tested, a restriction endonuclease and a 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 restriction endonuclease 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 double-stranded amplification product whose 5' end is the tag sequence and the restriction endonuclease specific recognition sequence; the restriction endonuclease will specifically recognize the restriction endonuclease specific recognition sequence in the double-stranded amplification product, and perform enzyme cleavage, respectively generating an enzyme cleavage incision in each double-stranded oligonucleotide chain; in the next round of PCR denaturation stage, the cut double-stranded oligonucleotide chain fragments are freed, and the fragments in the free fragments that can be complementary to the detection probe become reporter primers; the reporter primer can extend along the detection probe to form a new double-stranded oligonucleotide chain; Step 3: The reporter primer generated in step 2 is complementary paired and extended 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, thereby realizing the detection of the target nucleic acid to be detected.

[0029] Similarly, if Figure 3 and Figure 4 As shown, a method for detecting a target nucleic acid to be detected by melting curve is used for detecting a plurality of target nucleic acids to be detected, and the method comprises: Step 1: When multiple target nucleic acid sequences A1, A2...An are used as a group to simultaneously detect target nucleic acids, 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 comprises a tag sequence, a restriction endonuclease 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 restriction endonuclease specific recognition sequence refers to any single-stranded oligonucleotide chain sequence in the double-stranded oligonucleotide chain sequence recognized by the restriction endonuclease, 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 restriction endonuclease 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, 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 in the first primer B1 and a restriction endonuclease-specific recognition sequence; the detection probe is modified with a reporter group to form a melting curve peak in the detection stage; 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 restriction endonuclease and a DNA polymerase. If the sample to be tested contains multiple target nucleic acid sequences A1, A2...An to be tested, the target nucleic acid recognition sequences of the first primers B1, B2...Bn specifically bind to the corresponding target nucleic acids A1, A2...An to be tested, and extend 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 restriction endonuclease specific recognition sequence, the second primers C1, C2...Cn specifically bind to the single-stranded E1, E2...En generated by the extension, and extend thereon to generate double-stranded amplification products F1, F2...Fn whose 5' end is the tag sequence and the restriction endonuclease specific recognition sequence; the restriction endonuclease will specifically recognize the double-stranded amplification product F1, The restriction endonucleases in F2...Fn specifically recognize the sequences and perform enzyme cleavage, respectively generating an enzyme cleavage incision in the double-stranded oligonucleotide chain; in the next round of PCR denaturation stage, the cut double-stranded oligonucleotide chain fragments are freed, and the fragments in the free fragments that can be complementary to the detection probe become reporter primers G1, G2...Gn; the reporter primers can extend along the detection probe to form new double-stranded oligonucleotide chains; Step 3: The generated reporter primers G1, G2...Gn are respectively complementary paired and extended with the detection probe D to 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.

[0030] Example 2 EGFR single target detection experiment This embodiment takes the detection of EGFR as an example, and uses the method of the present invention to perform qualitative detection on the EGFR gene of human genomic DNA. The specific method includes the following steps: 1. Primer and probe sequence information The first primer, the second primer and the detection probe were designed according to the conserved region of the nucleic acid sequence to be detected. The sequence information is shown in the following table.

[0031] Table 1: Primer and probe sequences and concentrations used in this example

[0032] Note: Lowercase bases are tag sequences, lowercase bold letters are restriction endonuclease (BsiEI) specific recognition sequences, and uppercase letters are target nucleic acid recognition sequences 2. PCR Amplification Reaction System and Procedure The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis, wherein the PCR reaction system includes: 1× PCR buffer, 2 U BioZues® HS Multiplex Taq DNA Polymerase, 1 UBsiEI, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 100 nM detection probe, and 5 μL human genomic DNA.

[0033] The fluorescent PCR reaction program of the present invention is 95°C pre-denaturation for 1 min; 95°C denaturation for 5 s, 60°C annealing and extension for 45 s, collecting fluorescence at the same time, repeating 45 cycles; the melting curve program is 95°C for 2 min, 30°C for 5 min, 30-90°C for melting curve analysis, and detecting the fluorescence signal every 0.04°C. The instrument used in this embodiment is SLAN 96 real-time fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0034] 3. Test results The EGFR detection results of the method of the present invention are as follows Figure 5 As shown, the melting peak T m The value is 69.6℃. The baseline of this result is flat, the peak value is in line with expectations, and there is no non-specific peak.

[0035] Example 3 GAPDH single target detection experiment This embodiment takes the detection of GAPDH as an example, and uses the method of the present invention to perform qualitative detection on the GAPDH gene of human genomic DNA. The specific method includes the following steps: 1. Primer and probe sequence information The first primer, the second primer and the detection probe were designed according to the conserved region of the nucleic acid sequence to be detected. The sequence information is shown in the following table.

[0036] Table 2: Primer and probe sequences and concentrations used in the examples

[0037] Note: Lowercase bases are tag sequences, lowercase letters with underlines are SNP sites of tags, lowercase bold letters are restriction endonuclease (BsiEI) specific recognition sequences, and uppercase letters are target nucleic acid recognition sequences.

[0038] 2. PCR Amplification Reaction System and Procedure The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis, wherein the PCR reaction system includes: 1× PCR buffer, 2 U BioZues® HS Multiplex Taq DNA Polymerase, 1 U BsiEI, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 100 nM detection probe, and 5 μL human genomic DNA.

[0039] The fluorescent PCR reaction program of the present invention is 95°C pre-denaturation for 1 min; 95°C denaturation for 5 s, 60°C annealing and extension for 45 s, collecting fluorescence at the same time, repeating 45 cycles; the melting curve program is 95°C for 2 min, 30°C for 5 min, 30-90°C for melting curve analysis, and detecting the fluorescence signal every 0.04°C. The instrument used in this embodiment is SLAN 96 real-time fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0040] 3. Test results The GAPDH detection results of the method of the present invention are as follows Figure 6 As shown, the melting peak T m The value is 58.0℃. The baseline of this result is flat, the peak value is in line with expectations, and there is no non-specific peak.

[0041] Example 4 PIK3CA single target detection experiment This embodiment takes the detection of PIK3CA as an example, and uses the method of the present invention to perform qualitative detection on the PIK3CA gene of human genomic DNA. The specific method includes the following steps: 1. Primer and probe sequence information The first primer, the second primer and the detection probe were designed according to the conserved region of the nucleic acid sequence to be detected. The sequence information is shown in the following table.

[0042] Table 3: Primer and probe sequences and concentrations used in the examples

[0043] Note: Lowercase bases are tag sequences, lowercase letters with underlines are SNP sites of tags, lowercase bold letters are restriction endonuclease (BsiEI) specific recognition sequences, and uppercase letters are target nucleic acid recognition sequences.

[0044] 2. PCR Amplification Reaction System and Procedure The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis, wherein the PCR reaction system includes: 1× PCR buffer, 2 U BioZues® HS Multiplex Taq DNA Polymerase, 1 U BsiEI, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 100 nM detection probe, and 5 μL human genomic DNA.

[0045] The fluorescent PCR reaction program of the present invention is 95°C pre-denaturation for 1 min; 95°C denaturation for 5 s, 60°C annealing and extension for 45 s, collecting fluorescence at the same time, repeating 45 cycles; the melting curve program is 95°C for 2 min, 30°C for 5 min, 30-90°C for melting curve analysis, and detecting the fluorescence signal every 0.04°C. The instrument used in this embodiment is SLAN 96 real-time fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0046] 3. Test results The PIK3CA detection results of the method of the present invention are as follows Figure 7 As shown, the melting peak T m The value is 48.0℃. The baseline of this result is flat, the peak value is in line with expectations, and there is no non-specific peak.

[0047] Example 5 Triple Target Detection Experiment of the Present Invention This example takes the detection of gDNA as an example, and uses the method of the present invention to perform qualitative detection on human genomic DNA EGFR, GAPDH and PIK3CA genes. The specific method includes the following steps: 1. Primer and probe sequence information The first primer, the second primer and the detection probe were designed according to the conserved region of the nucleic acid sequence to be detected. The sequence information is shown in the following table.

[0048] Table 4: Primer and probe sequences and concentrations used in the examples

[0049] Note: Lowercase bases are tag sequences, lowercase letters with underlines are SNP sites of tags, lowercase bold letters are restriction endonuclease (BsiEI) specific recognition sequences, and uppercase letters are target nucleic acid recognition sequences.

[0050] 2. PCR Amplification Reaction System and Procedure The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis, wherein the PCR reaction system includes: 1× PCR buffer, 2 U BioZues® HS Multiplex Taq DNA Polymerase, 1 U BsiEI, 0.2 mM dNTPs, 40 nM of each first primer, 40 nM of each second primer, 300 nM of detection probe, and 5 μL of human genomic DNA.

[0051] The fluorescent PCR reaction program of the present invention is 95°C pre-denaturation for 1 min; 95°C denaturation for 5 s, 60°C annealing and extension for 45 s, collecting fluorescence at the same time, repeating 45 cycles; the melting curve program is 95°C for 2 min, 30°C for 5 min, 30-90°C for melting curve analysis, and detecting the fluorescence signal every 0.04°C. The instrument used in this embodiment is SLAN 96 real-time fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0052] 3. Test results The test results of genomic DNA EGFR, GAPDH, and PIK3CA are as follows Figure 8 As shown, the melting peak T m The values ​​are: 68.8℃, 57.6℃, 48.4℃ respectively. The baseline of this result is flat, 3 targets can be detected simultaneously, the peak value is in line with expectations, and there is no non-specific peak.

[0053] Example 6 Multiple target detection experiment of the present invention This embodiment takes the detection of gDNA as an example, and uses the method of the present invention to qualitatively detect the EGFR, GAPDH, PIK3CA, BRAF, ACTB, and CDA genes of human genomic DNA. The specific method includes the following steps: 1. Primer and probe sequence information The first primer, the second primer and the detection probe were designed according to the conserved region of the nucleic acid sequence to be detected. The sequence information is shown in the following table.

[0054] Table 5: Primer and probe sequences and concentrations used in the examples

[0055] Note: Lowercase bases are tag sequences, lowercase letters with underlines are SNP sites of tags, lowercase bold letters are restriction endonuclease (BsiEI) specific recognition sequences, and uppercase letters are target nucleic acid recognition sequences.

[0056] 2. PCR Amplification Reaction System and Procedure The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis, wherein the PCR reaction system includes: 1× PCR buffer, 2 U BioZues® HS Multiplex Taq DNA Polymerase, 1 U BsiEI, 0.2 mM dNTPs, 40 nM of each first primer, 40 nM of each second primer, 300 nM of each two detection probes, and 5 μL human genomic DNA.

[0057] The fluorescent PCR reaction program of the present invention is 95°C pre-denaturation for 1 min; 95°C denaturation for 5 s, 60°C annealing and extension for 45 s, collecting fluorescence at the same time, repeating 45 cycles; the melting curve program is 95°C for 2 min, 30°C for 5 min, 30-90°C for melting curve analysis, and detecting the fluorescence signal every 0.04°C. The instrument used in this embodiment is SLAN 96 real-time fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0058] 3. Test results The test results of genomic DNA EGFR, GAPDH, PIK3CA, BRAF, ACTB, CDA are as follows Fig. 9 As shown, the melting peak T of the FAM channel m The values ​​are: 69.2℃, 58℃, 47.8℃, ROX channel melting peak T m The values ​​are: 74℃, 65.6℃, 54.4℃ respectively. The baseline of this result is flat, 6 targets can be detected simultaneously, the peak value is in line with expectations, and there is no non-specific peak.

[0059] It should be understood that the disclosed invention is not limited only to the specific methods, schemes and materials described, because these can be varied. It should also be understood that the terms used herein are only for the purpose of describing specific implementation schemes, rather than being intended to limit the scope of the invention, which is limited only to the appended claims. Those skilled in the art will also recognize, or can confirm, using no more than routine experiments, many equivalents of the specific embodiments of the invention described herein. These equivalents are also included in the appended claims.

Claims

1. A method for detecting a target nucleic acid to be detected by 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 restriction endonuclease 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 restriction endonuclease specific recognition sequence refers to any single-stranded oligonucleotide chain sequence in the double-stranded oligonucleotide chain sequence recognized by the restriction endonuclease, 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 the tag sequence in the first primer and a restriction endonuclease-specific recognition sequence; the detection probe is modified with a reporter group to facilitate the formation of a melting curve peak in the detection stage; Step 2: PCR amplification is performed in a PCR amplification system containing the first primer, the second primer and the detection probe, the sample to be tested, a restriction endonuclease and a 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 restriction endonuclease 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 double-stranded amplification product whose 5' end is the tag sequence and the restriction endonuclease specific recognition sequence; the restriction endonuclease will specifically recognize the restriction endonuclease specific recognition sequence in the double-stranded amplification product, and perform enzyme cleavage, respectively generating an enzyme cleavage incision in each double-stranded oligonucleotide chain; in the next round of PCR denaturation stage, the cut double-stranded oligonucleotide chain fragments are freed, and the fragments in the free fragments that can be complementary to the detection probe become reporter primers; the reporter primer can extend along the detection probe to form a new double-stranded oligonucleotide chain; Step 3: The reporter primer generated in step 2 is complementary paired and extended 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, thereby realizing the detection of the target nucleic acid to be detected.

2. A method for detecting a target nucleic acid to be detected by 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 comprises a tag sequence, a restriction endonuclease 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 restriction endonuclease specific recognition sequence refers to any single-stranded oligonucleotide chain sequence in the double-stranded oligonucleotide chain sequence recognized by the restriction endonuclease, 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 restriction endonuclease 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, 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 in the first primer B1 and a restriction endonuclease-specific recognition sequence; the detection probe is modified with a reporter group to form a melting curve peak in the detection stage; 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 restriction endonuclease and a DNA polymerase. If the sample to be tested contains multiple target nucleic acid sequences A1, A2...An to be tested, the target nucleic acid recognition sequences of the first primers B1, B2...Bn specifically bind to the corresponding target nucleic acids A1, A2...An to be tested, and extend 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 restriction endonuclease specific recognition sequence, the second primers C1, C2...Cn specifically bind to the single-stranded E1, E2...En generated by the extension, and extend thereon to generate double-stranded amplification products F1, F2...Fn whose 5' end is the tag sequence and the restriction endonuclease specific recognition sequence; the restriction endonuclease will specifically recognize the double-stranded amplification product F1, The restriction endonucleases in F2...Fn specifically recognize the sequences and perform enzyme cleavage, respectively generating an enzyme cleavage incision in the double-stranded oligonucleotide chain; in the next round of PCR denaturation stage, the cut double-stranded oligonucleotide chain fragments are freed, and the fragments in the free fragments that can be complementary to the detection probe become reporter primers G1, G2...Gn; the reporter primers can extend along the detection probe to form a new double-stranded oligonucleotide chain; Step 3: The generated reporter primers G1, G2...Gn are respectively complementary paired and extended with the detection probe D to 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 restriction endonuclease 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 reporter group modified on the detection probe is a set of paired quencher group and fluorescent group.

7. The method for detecting a target nucleic acid by melting curve according to claim 1 or 2, characterized in that: The restriction endonuclease includes at least one of the following restriction endonucleases: ApeKI, ApoI, BclI, BfuAI, BsaBI, BsaJI, BsaWI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BsmBI-v2, BsmFI, BsmI, BspQI, BsrI, BssHII, BstAPI, BstBI, BstEII, BstNI, BstUI, BstYI, BtgZI, BtsCI, BtsIMutI, FatI, FauI, MwoI, PI-PspI, PspGI, SfiI, SmlI, TaqI-v2, TfiI, TseI, Tsp45I, TspMI, TspRI, Tth111I, EcoRI, EcoRV, HindIII, KpnI, XbaI, XhoI, and XmaI.

8. The method for detecting a target nucleic acid by melting curve according to claim 1 or 2, characterized in that: The DNA polymerase includes at least one of the following DNA polymerases: 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.

9. 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.

10. A kit for detecting a target nucleic acid to be detected by 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 restriction endonuclease 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 restriction endonuclease specific recognition sequence refers to any single-stranded oligonucleotide chain sequence in a double-stranded oligonucleotide chain sequence recognized by a restriction endonuclease, 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, the sequence of which completely comprises a continuous sequence consisting of the tag sequence in the first primer and a restriction endonuclease-specific recognition sequence; the detection probe is modified with a reporter group to form a melting curve peak during the detection stage.

11. A kit for detecting a target nucleic acid to be detected by 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 comprises a tag sequence, a restriction endonuclease 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 restriction endonuclease specific recognition sequence refers to any single-stranded oligonucleotide chain sequence in the double-stranded oligonucleotide chain sequence recognized by the restriction endonuclease, 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 restriction endonuclease 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, 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. Detection probe D, the sequence of which completely includes a continuous sequence consisting of the tag sequence in the first primer B1 and the restriction endonuclease specific recognition sequence; the detection probe is modified with a reporter group to form a melting curve peak in the detection stage.

12. 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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