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

Abstract

The present invention provides a method and a kit for detecting a target nucleic acid to be detected through 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 restriction endonuclease and a DNA polymerase, generating a reporter primer that is complementary to and pairs with 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 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 by melting curve and a kit thereof. 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. It has simple operation and wide application. However, this method only detects and analyzes in one dimension, that is, using the fluorescence signal generated by fluorescence change. 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 channel, generally only 1 to 6 target nucleic acids can be detected simultaneously 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, and use the generated Flaps fragment as an identification signal, and then perform 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 easy to cross-contaminate. 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, that is, fluorescence change and T m value, to achieve multiplex detection in one channel. The principle of the common melting curve analysis method is to use the T generated by the hybridization of a probe with a large number of single-stranded DNAs mThe melting curve analysis is performed on the values. 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 real-time fluorescence PCR multiplex detection and has the advantages of large 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, problems such as low amplification yield and low sensitivity are likely to occur. 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-stranded, hybrid double-stranded, and free single-stranded in the reaction. The fluorescence signal consists of two parts: from weak to strong and from strong to weak, and problems such as uneven baseline and inverted peaks are likely to occur. 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 double-strand. Different mediator primers and fluorescent molecular beacon probes extend to generate different fluorescent double-strands. During the melting curve analysis process, different fluorescent double-strands correspond to their respective m T values, and thus melting curve peaks are generated at different temperatures, thereby achieving 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 rises, 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 and 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 patents CN108823287B, CN110273012B, CN109988865B, and CN110273013B, there are the same problems 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 a 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 false positive background peaks generated by the binding of excessive mediator probes to the detection probe 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 by 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, wherein: a. The first primer is, from the 5'-end to the 3'-end, a tag sequence, a restriction endonuclease 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 restriction endonuclease specific recognition sequence refers to any one of the single-stranded oligonucleotide chain sequences in the double-stranded oligonucleotide chain sequence recognized by the restriction endonuclease, 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 contains the continuous sequence composed of the tag sequence and the restriction endonuclease 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 restriction endonuclease and the 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 restriction endonuclease specific recognition sequence of the first primer. The second primer specifically binds to the newly generated single strand and extends on it to generate a double-stranded amplification product with the 5'-end being the tag sequence and the restriction endonuclease specific recognition sequence; the restriction endonuclease specifically recognizes the restriction endonuclease specific recognition sequence in the double-stranded amplification product and performs enzymatic digestion, generating an enzymatic cleavage site in each of the double-stranded oligonucleotide chains; in the next round of PCR denaturation stage, the cleaved double-stranded oligonucleotide chain fragments are free, and the fragment that can be complementary to the detection probe in the free fragments becomes a reporter primer; the reporter primer can extend along the detection probe to form a new double-stranded oligonucleotide chain;

[0013] Step 3: The reporter primer generated in Step 2 is complementary paired with the detection probe and extends 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 by 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 is, from the 5'-end to the 3'-end, a tag sequence, a restriction endonuclease 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 restriction endonuclease specific recognition sequence refers to any one of the single-stranded oligonucleotide chain sequences in the double-stranded oligonucleotide chain sequence recognized by the restriction endonuclease, 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, and 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, and the tag sequences in the first primers B1, B2... Bn are different from each other. 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 corresponding 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;

[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 restriction endonuclease 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 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 among the target nucleic acid sequences 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 restriction endonuclease 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 tag sequences and the restriction endonuclease specific recognition sequences at their 5'-ends are generated by extension on them. The restriction endonuclease specifically recognizes the restriction endonuclease specific recognition sequences in the double-stranded amplification products F1, F2... Fn and performs enzymatic cleavage, generating an enzymatic cleavage site in each of the double-stranded oligonucleotide chains. In the denaturation stage of the next round of PCR, the cleaved double-stranded oligonucleotide chain fragments are released, and the fragments that can be complementary to the detection probe in the released fragments become reporter primers G1, G2... Gn. The reporter primers can extend along the detection probe to form new double-stranded oligonucleotide chains.

[0019] 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. 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 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 to be detected 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 progress.

[0022] In one embodiment, the detection probe sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, unnatural 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 restriction endonuclease 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 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 quenching groups include various commonly used quenching agents at present, but are not limited to these quenching agents, such as Dabcyl, Eclipse, BHQ-1, BHQ-2, QYS-7, etc.

[0025] In one embodiment, the restriction endonuclease includes at least one of the following restriction endonucleases: ApeKI (special buffer for non-inactivation), ApoI (special buffer for 80°C for 20 minutes), BclI (special buffer for non-inactivation), BfuAI (special buffer for 60°C for 20 minutes), 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, BtsIMutI, FatI, FauI, MwoI, PI-PspI, PspGI, SfiI, SmlI, TaqI-v2, TfiI, TseI, Tsp45I, TspMI, TspRI, Tth111I, EcoRI, EcoRV, HindIII, KpnI, XbaI, XhoI, XmaI.

[0026] In one embodiment, the DNA polymerase includes 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.

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

[0028] In one embodiment, a composition for detecting a target nucleic acid to be detected by melting curve is provided. The composition includes: a first primer, a second primer and a detection probe designed for the sequence of the target nucleic acid to be detected. Wherein, a. The first primer is, from the 5'-end to the 3'-end, a tag sequence, a restriction endonuclease 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 restriction endonuclease specific recognition sequence refers to any one single-stranded oligonucleotide chain sequence in the double-stranded oligonucleotide chain sequence recognized by the restriction endonuclease; the target nucleic acid recognition sequence refers to the sequence that specifically binds to the target nucleic acid to be detected;

[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 detected;

[0030] c. A detection probe, the sequence of which completely contains the continuous sequence composed of the tag sequence and the restriction endonuclease 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, a composition for detecting a target nucleic acid to be detected by melting curve is provided. The composition includes: first primers B1, B2... Bn, 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 are 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;

[0032] a. The first primer B1 is, from the 5'-end to the 3'-end, a tag sequence, a restriction endonuclease 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 restriction endonuclease specific recognition sequence refers to any one single-stranded oligonucleotide chain sequence in the double-stranded oligonucleotide chain sequence recognized by the restriction endonuclease; 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, and 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. The tag sequences in the first primers B1, B2... Bn are different from each other. 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 to facilitate the formation of melting curve peaks with different T m values;

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

[0034] c. A detection probe D, the sequence of which completely contains the continuous sequence composed of the tag sequence and the restriction endonuclease specific recognition sequence in the first primer B1; a reporter group is modified on the detection probe to facilitate the formation of 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 base paired and extended 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, 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 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 peak with the melting point (T m value) of a certain double-stranded body in the melting curve analysis, it can be judged whether the target nucleic acid sequence corresponding to the double-stranded body exists in the sample. This solves the problems of melting curve peak shift and easy misjudgment caused by the easy mutation of the target nucleic acid sequence to be detected, and well solves the problem that the melting curve analysis method is not applicable to RNA samples, greatly increasing the range of samples detected by the melting curve method and greatly improving the detection accuracy; at the same time, since the sequences of the reporting primer and the detection probe are artificially designed, known or predetermined, and the melting points of each double-stranded body are calculated in advance, the detection throughput can be greatly increased;

[0038] b. 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 extended thereon. The second primer specifically binds to the newly extended single strand and extends thereon to generate a double-stranded amplification product with the tag sequence and the specific recognition sequence of the restriction endonuclease at the 5' end. 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.

[0039] c. In the technical solution of the present invention, the reporting primer generated in step 2 is complementary paired and extended with the detection probe to form a double-stranded product, and the melting curve of this double-stranded product is obtained. The melting curve of this 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, and only the reporting primer can bind to the detection probe to generate a melting curve peak 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 well solves the problem of false positive background peaks and improves the detection accuracy and correct rate of interpretation.

[0040] d. 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.

[0041] e. 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 more prominently reflected, 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 per well and improving the detection throughput. With the method of the present invention, multiple target detections can be performed. 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 mThe value range is: 30~90 °C. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] 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 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.

[0043] Figure 1 Schematic diagram of the first primer used in the method of the present invention;

[0044] Figure 2 Schematic diagram of the basic principle of the method of the present invention;

[0045] Figure 3 Schematic diagram of multiple first primers for detecting multiple target nucleic acids to be detected by the method of the present invention;

[0046] Figure 4 Schematic diagram of the basic principle of detecting multiple target nucleic acids to be detected by the method of the present invention;

[0047] Figure 5 Schematic diagram of the detection result of EGFR by the method of the present invention;

[0048] Figure 6 Schematic diagram of the detection result of GAPDH by the method of the present invention;

[0049] Figure 7 Schematic diagram of the detection result of PIK3CA by the method of the present invention;

[0050] Figure 8 Schematic diagram of the triple detection result of EGFR, GAPDH, and PIK3CA by the method of the present invention

[0051] 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. SPECIFIC EMBODIMENTS

[0052] In order to enable those skilled in the art to 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 creative efforts shall fall within the scope of protection of the present application. In the following embodiments, unless otherwise specified, they are all conventional methods in the art.

[0053] Example 1 Basic Principle of the Present Invention

[0054] As Figure 1 and Figure 2 shown, the present invention provides a method for detecting a target nucleic acid by melting curve, a method for detecting a single target nucleic acid to be detected by melting curve, and the method includes:

[0055] A method for detecting a target nucleic acid to be detected by melting curve, and the method includes the following steps:

[0056] Step 1: Design a first primer, a second primer and a detection probe for the target nucleic acid sequence to be detected, wherein:

[0057] a. The first primer is, from the 5'-end to the 3'-end, a tag sequence, a restriction endonuclease 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 restriction endonuclease specific recognition sequence refers to any one of the single-stranded oligonucleotide chain sequences in the double-stranded oligonucleotide chain sequence recognized by the restriction endonuclease, and the target nucleic acid recognition sequence refers to the sequence specifically binding to the target nucleic acid to be detected;

[0058] b. A second primer, which 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;

[0059] c. A detection probe, the sequence of which contains the continuous sequence composed of the tag sequence and the restriction endonuclease 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;

[0060] 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 restriction endonuclease and the 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 thereon to generate a new single strand, the 5'-end of which 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 extended above and extends thereon to generate a double-stranded amplification product with the 5'-end being the tag sequence and the restriction endonuclease specific recognition sequence; the restriction endonuclease specifically recognizes the restriction endonuclease specific recognition sequence in the double-stranded amplification product and performs enzymatic digestion, generating an enzymatic cleavage site in each of the double-stranded oligonucleotide chains. In the next round of PCR denaturation stage, the cleaved double-stranded oligonucleotide chain fragments are free, and the fragment that can be complementary to the detection probe in the free fragments becomes a reporter primer; the reporter primer can extend along the detection probe to form a new double-stranded oligonucleotide chain;

[0061] Step 3: The reporting primer generated in Step 2 is complementary to and extends 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.

[0062] Similarly, as Figure 3 and Figure 4 shown, a method for detecting a target nucleic acid to be detected by melting curve, the method is used for detecting multiple target nucleic acids to be detected, and the method includes:

[0063] Step 1: When multiple target nucleic acid sequences A1, A2... An are detected as a group simultaneously for the target nucleic acid to be detected, corresponding first primers B1, B2... Bn and second primers C1, C2... Cn and a common detection probe D are respectively designed for each of the target nucleic acid sequences A1, A2... An, where n is an integer not less than 2;

[0064] a. The first primer B1 is, from the 5'-end to the 3'-end, in sequence a tag sequence, a restriction endonuclease specific recognition sequence, and a target nucleic acid recognition sequence; the tag sequence cannot bind to the target nucleic acid to be detected, the restriction endonuclease specific recognition sequence refers to any one of the single-stranded oligonucleotide chain sequences in the double-stranded oligonucleotide chain sequence recognized by the restriction endonuclease, 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 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, 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;

[0065] 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 and the corresponding first primers B1, B2... Bn are respectively used in combination to amplify the corresponding target nucleic acid sequences A1, A2... An;

[0066] c. Detection probe D, the sequence of the detection probe D completely contains the continuous sequence composed of the tag sequence and the restriction endonuclease specific recognition sequence in the first primer B1; a reporter group is modified on the detection probe, so as to facilitate the formation of melting curve peaks in the detection stage;

[0067] 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 restriction endonuclease, 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 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 restriction endonuclease 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 tag sequences and the restriction endonuclease specific recognition sequences at their 5'-ends are generated by extension on them. The restriction endonuclease specifically recognizes the restriction endonuclease specific recognition sequences in the double-stranded amplification products F1, F2... Fn and performs enzymatic digestion, generating an enzymatic cleavage site in each of the double-stranded oligonucleotide chains respectively. In the denaturation stage of the next round of PCR, the cleaved double-stranded oligonucleotide chain fragments are released, and the fragments that can be complementary to the detection probe in the released fragments become reporter primers G1, G2... Gn. The reporter primers can extend along the detection probe to form new double-stranded oligonucleotide chains.

[0068] 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. 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.

[0069] Example 2: EGFR single-target detection experiment

[0070] 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:

[0071] I. Primer and probe sequence information

[0072] 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.

[0073] Table 1: Primer, probe sequences and usage concentrations involved in this example

[0074]

[0075] Note: The lowercase letter bases are the tag sequences, the bold lowercase letters are the specific recognition sequences of the restriction endonuclease (BsiEI), and the uppercase letters are the target nucleic acid recognition sequences.

[0076] II. PCR Amplification Reaction System and Procedure

[0077] 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 BsiEI, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 100 nM detection probe, and 5 μL human genomic DNA.

[0078] 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.).

[0079] III. Detection Results

[0080] The EGFR detection result of the method of the present invention is as Figure 5 shown, the melting peak T m value is at 69.6°C. The baseline of this result is flat, the peak value meets expectations, and there are no non-specific peaks.

[0081] Example 3 GAPDH Single Target Detection Experiment

[0082] Taking the detection of GAPDH as an example, this example qualitatively detects the GAPDH gene of human genomic DNA by the method of the present invention. The specific method includes the following steps:

[0083] I. Primer and Probe Sequence Information

[0084] 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.

[0085] Table 2: Primer and Probe Sequences and Usage Concentrations Involved in the Example

[0086]

[0087] 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 restriction endonuclease (BsiEI), and the uppercase letters are the target nucleic acid recognition sequences.

[0088] II. PCR Amplification Reaction System and Program

[0089] 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 BsiEI, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 100 nM detection probe, and 5 μL human genomic DNA.

[0090] The fluorescence PCR reaction program 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 program 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.).

[0091] III. Detection Results

[0092] 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 58.0°C. The baseline of this result is flat, the peak value meets the expectation, and there are no non-specific peaks.

[0093] Example 4 PIK3CA Single-Target Detection Experiment

[0094] 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:

[0095] I. Primer and Probe Sequence Information

[0096] The first primer, the second primer, and the 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.

[0097] Table 3: Primer and Probe Sequences and Usage Concentrations Involved in the Example

[0098]

[0099] 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 restriction endonuclease (BsiEI), and the uppercase letters are the target nucleic acid recognition sequences.

[0100] II. PCR Amplification Reaction System and Procedure

[0101] 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 BsiEI, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 100 nM detection probe, and 5 μL human genomic DNA.

[0102] 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.).

[0103] III. Detection Results

[0104] 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 48.0°C. The baseline of this result is flat, the peak value meets the expectation, and there are no non-specific peaks.

[0105] Example 5 Triple Target Detection Experiment of the Present Invention

[0106] 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:

[0107] I. Primer and Probe Sequence Information

[0108] 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.

[0109] Table 4: Primer and Probe Sequences and Usage Concentrations Involved in the Example

[0110]

[0111] 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 restriction endonuclease (BsiEI), and the uppercase letters are the target nucleic acid recognition sequences.

[0112] II. PCR Amplification Reaction System and Program

[0113] 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 BsiEI, 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.

[0114] The fluorescence PCR reaction program 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 program 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.).

[0115] III. Detection Results

[0116] The detection results of genomic DNA EGFR, GAPDH, and PIK3CA are as Figure 8 shown. The melting peak T m values are respectively: 68.8°C, 57.6°C, 48.4°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.

[0117] Example VI Multiple Target Detection Experiment of the Present Invention

[0118] 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:

[0119] I. Primer and Probe Sequence Information

[0120] 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.

[0121] Table 5: Primer and Probe Sequences and Usage Concentrations Involved in the Example

[0122]

[0123] 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 restriction endonuclease (BsiEI), and the capital letters are the target nucleic acid recognition sequences.

[0124] II. PCR Amplification Reaction System and Procedure

[0125] 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 BsiEI, 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.

[0126] 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.).

[0127] III. Detection Results

[0128] The detection results of genomic DNA for EGFR, GAPDH, PIK3CA, BRAF, ACTB, and CDA are as Figure 9 shown. The melting peak T m values in the FAM channel are: 69.2°C, 58°C, 47.8°C, and the melting peak T m values in the ROX channel are: 74°C, 65.6°C, 54.4°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.

[0129] 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 for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Those skilled in the art will also recognize, or be able to confirm using no more than routine experimentation, many equivalents of the specific embodiments of the present 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 form a melting curve peak in the detection stage, and the reporter group modified on the detection probe is a set of paired quenching groups and fluorescent groups; 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 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, and the reporter group modified on the detection probe is a pair of a quencher group and a fluorescent group; 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 by melting curve analysis 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 by melting curve analysis 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 by melting curve analysis 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 by melting curve analysis 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.

7. The method for detecting a target nucleic acid by melting curve analysis 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, 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 by melting curve analysis 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 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, wherein the sequence of the detection probe completely includes a continuous sequence consisting of the label 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 phase, and 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 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. A detection probe D, wherein the sequence of the detection probe D completely comprises a continuous sequence consisting of the label 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 during the detection phase, and the reporter group modified on the detection probe 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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