A probe and primer for detecting nucleic acid variation based on melting curve method, and method

Abstract

The present invention relates to the field of biotechnology, and specifically discloses a probe and primer for detecting nucleic acid variation based on a melting curve method, and a method. A probe and primer for detecting nucleic acid variation based on a melting curve method, the primer consists of an upstream primer and a downstream primer; the probe is a hairpin structure probe, and from the 5' end to the 3' end, there are a stem, a loop and a stem in sequence, the two stems are complementary sequences, and the sequence of the loop is the same as or complementary to the sequence of wild-type DNA; the 3' end of the probe has a modification that can block polymerase extension, and the partial sequence of the probe close to the 5' end overlaps with the partial sequence starting from the 3' end of the upstream primer. A method for detecting nucleic acid variation based on a melting curve method, using the probe and primer prepared by the present application. The detection method of the present application improves the detection ability of the PCR melting curve method for low-abundance variant DNA, and can detect variant DNA with a mutation frequency of less than 1% at the lowest.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a probe and primer for detecting nucleic acid variation based on a melting curve method, and a method. Background Art

[0002] The PCR melting curve is a curve of the change in fluorescence intensity obtained by the change in the fluorescence signal related to the PCR product during the heating process of the PCR product. During the heating process, the temperature corresponding to the half-melting of the DNA is called the melting temperature. The melting temperature is related to the sequence composition and length of the DNA double helix. In the case of similar sequences, the difference of a few or even a single nucleotide sequence may also cause a significant difference in its melting temperature, resulting in a difference in the melting curve. This is the basic principle of using the melting curve method to distinguish different nucleic acid sequences, such as SNP, different pathogens and even DNA mutations.

[0003] The traditional PCR melting curve method is mainly divided into two types: 1) Based on fluorescent dyes such as SYBR green I. The principle is that the fluorescent dye can bind to the minor groove of double-stranded DNA. As the temperature rises, the DNA double strands gradually melt into single strands, the fluorescent dye falls off into a free state, and the signal weakens. The change in the intensity of the fluorescent signal in this process forms a melting curve. 2) Based on fluorescent probes. In such a design, one end of the probe is connected to a fluorescent group and the other end is connected to a quenching group. When the probe is in a free state, the fluorescent group and the quenching group are close to each other, and the quenching group completely absorbs the fluorescence of the fluorescent group, resulting in no fluorescent signal. When the probe forms a double strand with the target nucleic acid, the probe is in a stretched state, the fluorescent group and the quenching group are far away, and the fluorescent group emits fluorescence.

[0004] At present, the PCR melting curve method is mainly used for the detection of SNPs and pathogens. In these application scenarios, some test samples have a large difference in the number of DNA / RNA molecules with differential sequences. For example, for SNP, the proportion of DNA molecules with differential sequences may be 0%, 50% and 100%. Due to the large difference in the number of such test samples, the amount of different PCR products varies greatly. Due to the difference in the melting temperature of different PCR products, the difference in melting curves can be clearly seen, so different SNPs can be typed. For other applications, such as the detection of somatic DNA mutations in tumor tissues, the traditional PCR melting curve method has some defects: due to the heterogeneity of tumor tissue cells, DNA mutations may only occur in a part of the cells, so the proportion of mutant DNA may be as low as 1% or less, which puts higher requirements on the PCR melting curve method. Existing studies have shown that the traditional melting curve method based on fluorescent probes can stably detect 1% to 10% of mutant DNA at the lowest, but due to the limitations of the technology itself, it is difficult to detect mutant DNA below 1%.

[0005] Therefore, in order to improve the sensitivity and accuracy of somatic DNA variation detection in tumor tissues, it is necessary to further improve the traditional PCR melting curve method and enhance the detection ability of the PCR melting curve method for low-abundance variant DNA. Summary of the invention

[0006] In order to improve the sensitivity and accuracy of somatic DNA variation detection in tumor tissues, the present application provides a probe and primer for detecting nucleic acid variation based on the melting curve method. The detection method of the present application improves the detection ability of the PCR melting curve method for low-abundance variant DNA, and can detect variant DNA with a mutation frequency of less than 1%.

[0007] In the first aspect, the present application provides a probe and primer for detecting nucleic acid variation based on the melting curve method using the following technical solution:

[0008] A probe and primer for detecting nucleic acid variation based on a melting curve method, wherein the primer consists of an upstream primer and a downstream primer, the Tm value of the upstream primer is less than the Tm value of the probe; the probe is a hairpin structure probe, and from the 5' end to the 3' end, there are a stem, a loop and a stem in sequence, the length of the stem is 4-8 bp, the two stems are complementary sequences, the length of the loop is 13-40 bp, and the sequence of the loop is the same as or complementary to that of a wild-type DNA; the 3' end of the probe has a modification that can block polymerase extension, and the partial sequence of the probe close to the 5' end overlaps with the partial sequence starting from the 3' end of the upstream primer.

[0009] Preferably, the modification used at the 3' end of the probe that can block polymerase extension includes but is not limited to 3'ddC, 3'Inverted dT, 3'C3 spacer, 3'amino, 3'phosphorylation and quencher group.

[0010] Preferably, the quenching group includes but is not limited to BHQ1, BHQ2, BHQ3, TAMARA and Dabcyl.

[0011] Preferably, the probe is a fluorescent modified probe, and a fluorescent group is connected to the 5' end of the fluorescent modified probe.

[0012] Preferably, the fluorescent group includes but is not limited to FAM, TET, JOE, VIC, HEX, NED, ROX, Tamara, Cy3 and Cy5.

[0013] Preferably, the length of the overlapping sequence is 4 to 12 bp.

[0014] Preferably, the probe is as shown in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, and SEQ ID NO:15, the upstream primer is as shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:13, and the downstream primer is as shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, and SEQ ID NO:14.

[0015] In a second aspect, the present application provides a method for detecting nucleic acid variation based on a melting curve method using the following technical solution:

[0016] A method for detecting nucleic acid variation based on a melting curve method, using the probe and primers described in the first aspect.

[0017] Preferably, a method for detecting nucleic acid variation based on a melting curve method comprises the following steps:

[0018] Step 1, design probes and primers;

[0019] Step 2, configuration of PCR reaction system: such as formula 1 or formula 2;

[0020] The formula 1 consists of a buffer, dNTPs, an upstream primer, a downstream primer, a fluorescent modified probe, a DNA polymerase, a DNA template and nuclease-free water;

[0021] Formula 2 consists of a buffer, dNTPs, an upstream primer, a downstream primer, a probe without fluorescence modification, a fluorescent dye, a DNA polymerase, a DNA template, and nuclease-free water;

[0022] Step 3, PCR amplification reaction;

[0023] The PCR amplification reaction procedure comprises: the first step: pre-denaturation, 90-95°C, 7-8min, 1 cycle in total; the second step: amplification, 90-95°C, 10-60s, 50-72°C, 30-120s, 40-60 cycles in total; the third step: denaturation, 90-95°C, 2-3min, 1 cycle in total; the fourth step: renaturation, 25-45°C, 1-2min, 1 cycle in total;

[0024] Step 4, melting curve detection: heating range 25-90°C, heating rate 0.02-0.4°C / s, a total of 1 cycle;

[0025] Step 5: Analysis of melting curve detection results.

[0026] Preferably, the formula 1 consists of 1x buffer, 0.2~0.4μM dNTPs, 0.05~0.2μM upstream primer, 1~2μM downstream primer, 0.1~2μM fluorescent modified probe, DNA polymerase, 0.05~0.1U / μL DNA template and an appropriate amount of nuclease-free water.

[0027] Preferably, the formula 2 consists of 1x buffer, 0.2~0.4μM dNTPs, 0.05~0.2μM upstream primer, 1~2μM downstream primer, 0.1~2μM non-fluorescent modified probe, 1x fluorescent dye, 0.05~0.1U / μL DNA polymerase, DNA template and an appropriate amount of nuclease-free water.

[0028] Preferably, the melting curve detection results are analyzed as follows:

[0029] Use formula 1, that is, the PCR reaction system using fluorescent modified probes:

[0030] When the DNA template used as the test sample contains mutant DNA, in the analysis of the melting curve test results, the melting curve of the test group containing mutant DNA will have at least one melting peak, compared with the wild-type control group which has only one or no melting peak. Among them, the melting temperature of the probe after binding to DNA with single-site or multi-site mutations is lower, while the melting temperature of the probe after binding to non-mutated DNA is higher or there is no melting peak (at this time, the amplification of wild-type DNA is completely suppressed). The temperatures corresponding to the melting peaks of the two can be clearly distinguished, thereby achieving the purpose of detecting mutant DNA.

[0031] When the DNA template used as the test sample does not contain variant DNA, in the analysis of the melting curve detection results, the melting curve only produces one or no melting peak. When a melting peak exists, its corresponding temperature is higher, and the temperature corresponding to the variant DNA melting peak can be clearly distinguished.

[0032] Use formula 2, which is a PCR reaction system using fluorescent dyes:

[0033] When using fluorescent dyes, the double-stranded DNA of the PCR product itself will produce a melting peak when it melts. Since the PCR product is long, the temperature corresponding to the melting peak is higher. At the same time, the probe that is not fluorescently labeled will also form a DNA double-strand with the PCR product, which will produce another melting peak when it melts. Since the double-stranded DNA is short, the temperature corresponding to the melting peak is lower. Therefore, whether it is wild-type or variant DNA, its PCR product will have two melting peaks. For the melting peak of the double-stranded DNA of the PCR product itself, if the degree of base variation is small, such as point mutation, the difference between the melting peaks of the PCR products of wild-type DNA and variant DNA may be small, but at the same time, the temperature corresponding to the melting peak formed by the PCR products of wild-type DNA and variant DNA and the probe is quite different. Among them, the melting temperature of the probe and the wild-type DNA product will be greater than that of the variant DNA product, and the difference can be used to detect the variant DNA.

[0034] Preferably, the fluorescent dye includes but is not limited to SYBR green I, EvaGreen and LCgreen.

[0035] In summary, the present application includes at least one of the following beneficial technical effects:

[0036] The present application requires that there must be an overlapping portion between the upstream primer and the probe, that there is a Tm (melting temperature) difference between the probe and the upstream primer, that there is a Tm difference between the probe and the wild-type and mutant DNAs, and that the sequence of the loop of the probe is identical to or complementary to the sequence of the wild-type DNA. Through the above settings, since the probe perfectly matches the wild-type DNA and has a strong binding force, competition is generated when the upstream primer and the probe bind to the wild-type DNA, and the probe can well suppress the binding of the wild-type DNA and the upstream primer, thereby suppressing or even completely suppressing the amplification of the wild-type DNA, while the mutant DNA does not perfectly match the probe and has a weak binding force, so the upstream primer can better bind to and extend the amplification of the mutant DNA. Therefore, at a suitable annealing temperature, the probe binds to the wild-type DNA but not to the mutant DNA, while the upstream primer binds more to the mutant DNA, thereby preferentially amplifying the mutant DNA, thereby achieving significant amplification of the mutant DNA, thereby further improving the detection ability of the PCR melting curve method for low-abundance variant DNA. Using the detection method of the present application, the variant DNA with a mutation frequency of less than 1% can be detected at the lowest. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : is a schematic diagram illustrating the method for detecting nucleic acid variation based on the melting curve method of the present application, wherein: Figure 1 A in FIG. 1 is a schematic diagram of the structure of the hairpin probe of the present application; Figure 1 B is a schematic diagram of the hairpin probe of the present application preventing amplification of wild-type DNA; Figure 1C in the figure is a schematic diagram of the melting curve detection results of the mutant DNA detected in the present application.

[0038] Figure 2 It is a melting curve detection diagram of Example 1 of the present application, wherein, marked wild type: melting peak formed by PCR amplification product of wild-type DNA in Example 1 and probe; marked 0.2%: melting peak formed by PCR amplification product of mutant DNA with 0.2% mutation frequency in Example 1 and probe; marked 0.5%: melting peak formed by PCR amplification product of mutant DNA with 0.5% mutation frequency in Example 1 and probe; marked 1%: melting peak formed by PCR amplification product of mutant DNA with 1% mutation frequency in Example 1 and probe; marked 2%: melting peak formed by PCR amplification product of mutant DNA with 2% mutation frequency in Example 1 and probe, melting curve marked NTC: melting curve of control sample without DNA template.

[0039] Figure 3 It is a melting curve detection diagram of Example 2 of the present application, wherein the wild type is marked: the melting peak formed by the PCR amplification product of the wild type DNA of Example 2 and the probe; the 0.5% is marked: the melting peak formed by the PCR amplification product of the mutant DNA with a mutation frequency of 0.5% and the probe of Example 2; the 1% is marked: the melting peak formed by the PCR amplification product of the mutant DNA with a mutation frequency of 1% and the probe of Example 2; the 2% is marked: the melting peak formed by the PCR amplification product of the mutant DNA with a mutation frequency of 2% and the probe of Example 2; the melting curve marked with NTC: the melting curve of the control sample without DNA template.

[0040] Figure 4 It is the melting curve detection diagram of Example 3 of the present application, wherein the melting peak formed by the wild-type PCR amplification product and the probe is marked: the melting peak formed by the PCR amplification product of the wild-type DNA in Example 3 and the probe; the melting peak of the wild-type PCR product is marked: the melting peak formed by the PCR amplification product of the wild-type DNA in Example 3; the melting peak formed by the mutant PCR amplification product and the probe is marked: the melting peak formed by the PCR amplification product of the mutant DNA with a mutation frequency of 0.2% in Example 3 and the probe; the melting peak of the mutant PCR product is marked: the melting peak formed by the PCR amplification product of the mutant DNA with a mutation frequency of 0.2% in Example 3; the melting curve marked with NTC: the melting curve of the control sample without DNA template.

[0041] Figure 5It is a melting curve detection diagram of Example 4 of the present application, wherein the wild type is marked: the melting peak formed by the PCR amplification product of the wild type DNA of Example 4 and the probe; the 0.2% is marked: the melting peak formed by the PCR amplification product of the mutant DNA with a mutation frequency of 0.2% and the probe of Example 4; the 0.5% is marked: the melting peak formed by the PCR amplification product of the mutant DNA with a mutation frequency of 0.5% and the probe of Example 4; the 1% is marked: the melting peak formed by the PCR amplification product of the mutant DNA with a mutation frequency of 1% and the probe of Example 4; the melting curve marked with NTC: the melting curve of the control sample without DNA template.

[0042] Figure 6 It is a melting curve detection diagram of Example 5 of the present application, wherein the wild type is marked: the melting peak formed by the PCR amplification product of the wild type DNA in Example 5 and the probe; the 0.5% is marked: the melting peak formed by the PCR amplification product of the mutant DNA with a mutation frequency of 0.5% in Example 5 and the probe; the 1% is marked: the melting peak formed by the PCR amplification product of the mutant DNA with a mutation frequency of 1% in Example 5 and the probe; the 2% is marked: the melting peak formed by the PCR amplification product of the mutant DNA with a mutation frequency of 2% in Example 5 and the probe; the melting curve marked with NTC: the melting curve of the control sample without DNA template. DETAILED DESCRIPTION

[0043] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Embodiments of the present invention are described in detail below, and the embodiments described are exemplary and are intended to be used to explain the present invention, but should not be construed as limitations of the present invention. If specific techniques or conditions are not indicated in the embodiments, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specification. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0044] The primers of the present application include upstream primers and downstream primers.

[0045] Among them, the length of the upstream primer of the present application is adjusted according to the requirements of the Tm value in the PCR primer design, while ensuring that the Tm value of the upstream primer is less than the Tm value of the probe.

[0046] Among them, the length of the downstream primer of the present application is adjusted according to the requirements of the Tm value in the PCR primer design, and can be 20bp, 21bp, 22bp, 23bp, 24bp, 25bp, 26bp, 27bp, 28bp, 29bp, 30bp, 31bp or 32bp.

[0047] like Figure 1As shown in A in FIG. 1 , the probe of the present application has a hairpin structure, consisting of two stems and a loop.

[0048] Among them, the two stems are complementary sequences.

[0049] The length of a stem may be 4 bp, 5 bp, 6 bp, 7 bp or 8 bp.

[0050] The sequence of the loop is identical to or complementary to the sequence of the wild-type DNA.

[0051] Among them, the length of the loop can be 13bp, 14bp, 15bp, 16bp, 17bp, 18bp, 19bp, 20bp, 21bp, 22bp, 23bp, 24bp, 25bp, 26bp, 27bp, 28bp, 29bp, 30bp, 31bp, 32bp, 33bp, 34bp, 35bp, 36bp, 37bp, 38bp, 39bp or 40bp.

[0052] The 3' end of the probe of the present application is modified to block polymerase extension.

[0053] The modification that can block polymerase extension can be 3'ddC, 3'Inverted dT, 3'C3 spacer, 3'amino, 3'phosphorylation or a quencher group.

[0054] The quenching group may be BHQ1, BHQ2, BHQ3, TAMARA or Dabcyl.

[0055] The 5' end of the probe of the present application may be fluorescently modified, that is, a fluorescent group may be connected to the 5' end.

[0056] Among them, the fluorescent group can be FAM, TET, JOE, VIC, HEX, NED, ROX, Tamara, Cy3 or Cy5.

[0057] The 5' end of the probe of the present application may not be fluorescently modified. In this case, a fluorescent dye needs to be added to the PCR reaction system used in the detection of the present application.

[0058] The fluorescent dye may be SYBR green I, EvaGreen or LCgreen.

[0059] The partial sequence near the 5' end of the probe of the present application overlaps with the partial sequence starting from the 3' end of the upstream primer. The overlapping sequence is the overlapping sequence, and the length of the overlapping sequence is adjusted according to the effect of suppressing the amplification of the wild-type DNA.

[0060] The length of the overlapping sequence may be 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 11 bp or 12 bp.

[0061] Among them, in the probe sequence of the present application, all overlapping sequences are located on the loop structure or 4 to 8 bp are located on the loop structure and 1 to 4 bp are located on the stem structure.

[0062] like Figure 1 As shown in B, during the PCR amplification reaction, during annealing, the stem loop of the probe opens, and the loop sequence of the probe is identical or complementary to the sequence of the wild-type DNA. The probe binds to the wild-type DNA. At this time, the probe occupies the position on the wild-type DNA that should be bound to the primer, resulting in the inability of the primer to bind to the wild-type DNA and to extend to produce products, thereby suppressing the amplification of the wild-type DNA. At the same time, since the loop sequence of the probe cannot be 100% matched with the sequence of the mutant DNA, during annealing, the probe cannot stably bind to the mutant DNA, and the position on the mutant DNA that binds to the primer is vacant, and the primer can bind to the mutant DNA, thereby extending to produce mutant DNA amplification products.

[0063] In addition, since the partial sequence of the probe of the present application near the 5' end overlaps with the partial sequence starting from the 3' end of the upstream primer, the overlap between the upstream primer and the probe causes competition between the two when binding to the template. Since the probe perfectly matches the wild-type DNA and has a strong binding force, the amplification of the wild-type DNA can be suppressed, so that only part of the wild-type DNA can bind to the upstream primer to produce amplification, or no wild-type DNA binds to the upstream primer, completely suppressing the amplification of the wild-type DNA.

[0064] like Figure 1 As shown in C in the figure, in the analysis of the melting curve detection results, the melting temperature of the probe after binding to the mutant DNA product is lower, which can clearly distinguish the melting temperature generated by its binding to the wild-type DNA product, thereby achieving the purpose of detecting mutant DNA.

[0065] Example 1

[0066] Detection of KRAS G12C site mutation

[0067] Step 1: Design primers and probes:

[0068] Upstream primer: 5'-TCAAGGCACTCTTGCCTAC-3' (SEQ ID NO: 1)

[0069] Downstream primer: 5'-AGGCCTGCTGAAAATGACTG-3' (SEQ ID NO: 2)

[0070] Probe: FAM-5'-TGGA GC CTAC GCCACCA GCTCCA -3'-BHQ1 (the middle sequence is SEQ ID NO: 3)

[0071] The single underline mark of the probe refers to the complementary sequence of the hairpin structure, the italic mark refers to the overlapping portion of the probe and the upstream primer sequence, and the bold mark refers to the mutation site of the mutant DNA in this example.

[0072] Step 2: The preparation of PCR reaction system is shown in Table 1.

[0073] Table 1:

[0074]

[0075] Step 3: Referring to the reaction system in step 2, use 0.2%, 0.5%, 1%, 2% mutation frequency DNA and wild-type DNA as DNA templates to prepare one reaction each, and perform the reaction in a Macrostone 96S qPCR instrument according to the reaction program in Table 2 below.

[0076] Table 2:

[0077]

[0078] Step 4: Observation of results: See the test results Figure 2 .

[0079] from Figure 2 It can be seen from the test results that the melting temperature of the melting peak formed by the PCR amplification product of wild-type DNA and the probe is around 63°C, while for mutant DNA with mutation frequencies of KRAS G12C 0.2%, 0.5%, 1%, and 2%, in addition to the melting peak at around 63°C, there is also a melting peak formed by the PCR amplification product of mutant DNA and the probe at a lower temperature of around 52°C, which indicates that the detection method of the present application can achieve the purpose of detecting variant DNA through the feature of inconsistent melting peaks, and can effectively distinguish wild-type DNA from mutant DNA. At the same time, this embodiment also shows that the detection method of the present application can at least detect DNA mutations with a mutation frequency of 0.2%-2%.

[0080] Example 2

[0081] Detection of KRAS A146T site mutation

[0082] Step 1: Design primers and probes:

[0083] Upstream primer: 5'-AGTGTTACTTACCTGTCTTGTCTTT-3' (SEQ ID NO: 4)

[0084] Downstream primer: 5'-GAAGTTATGGAATTCCTTTTATTGAAACATCA-3' (SEQ ID NO: 5)

[0085] Probe: VIC-5'- ccaa C TTGTCTT GC】TGAT GTT -3'-BHQ1 (the middle sequence is SEQ ID NO: 6)

[0086] Among them, the [ ] in the probe is an LNA modified base, the single underline mark refers to the complementary sequence of the hairpin structure, the italic mark refers to the overlapping part of the probe and the upstream primer sequence, and the bold mark refers to the mutation site of the mutant DNA in this example.

[0087] Step 2: The preparation of PCR reaction system is shown in Table 3.

[0088] Table 3:

[0089]

[0090] Step 3: Referring to the reaction system in step 2, use 0.5%, 1%, 2% mutation frequency DNA and wild-type DNA as DNA templates to prepare one reaction each, and perform the reaction in a Macrostone 96S qPCR instrument according to the reaction program in Table 4 below.

[0091] Table 4:

[0092]

[0093] Step 4: Observation of results: See the test results Figure 3 .

[0094] from Figure 3 It can be seen from the test results that the melting temperature of the melting peak formed by the PCR amplification product of wild-type DNA and the probe is around 62°C, while for mutant DNA with mutation frequencies of KRAS A46T 0.5%, 1%, and 2%, in addition to the melting peak at around 62°C, there is also a melting peak formed by the PCR amplification product of mutant DNA and the probe at a lower temperature of around 50°C, which indicates that the detection method of the present application can achieve the purpose of detecting variant DNA through the feature of inconsistent melting peaks, and can effectively distinguish wild-type DNA from mutant DNA. At the same time, this embodiment also shows that the detection method of the present application can at least detect DNA mutations with a mutation frequency of 0.5%-2%.

[0095] Example 3

[0096] Detection of KRAS A146V site mutation

[0097] Step 1: Design primers and probes:

[0098] Upstream primer: 5'-AGTGTTACTTACCTGTCTTGTCTTT-3' (SEQ ID NO: 7)

[0099] Downstream primer: 5'-GAAGTTATGGAATTCCTTTTATTGAAACATCA-3' (SEQ ID NO: 8)

[0100] Probe: 5'- ccaa C TTGTCTT GC】TGAT GTT -3'-spC3 (the middle sequence is SEQ ID NO: 9)

[0101] Among them, the [ ] in the probe is an LNA modified base, the single underline mark refers to the complementary sequence of the hairpin structure, the italic mark refers to the overlapping part of the probe and the upstream primer sequence, and the bold mark refers to the mutation site of the mutant DNA in this example.

[0102] Step 2: The PCR reaction system is prepared as shown in Table 5.

[0103] Table 5:

[0104]

[0105] Step 3: Referring to the reaction system in step 2, use 0.2% mutation frequency DNA and wild-type DNA as DNA templates to prepare one reaction each, and perform the reaction in a Macrostone 96S qPCR instrument according to the reaction program in Table 6 below.

[0106] Table 6:

[0107]

[0108] Step 4: Observation of results: See the test results Figure 4 .

[0109] from Figure 4 It can be seen from the test results that, because this example does not use fluorescently modified probes, but directly adds fluorescent dyes to the PCR reaction system, compared with the detection method using fluorescently modified probes, there are additional wild-type PCR product melting peaks and mutant PCR product melting peaks. However, for the analysis of the test results, only the melting peak formed by the wild-type PCR amplification product and the probe and the melting peak formed by the PCR amplification product of the mutant DNA with a mutation frequency of 0.2% and the probe are considered. Specific observation Figure 4The melting temperature of the melting peak formed by the wild-type PCR amplification product and the probe is about 78°C, while for the mutant DNA with a mutation frequency of KRAS G12V0.2%, the melting temperature of the melting peak formed by the PCR product and the probe is about 72°C. There is a significant difference in the melting temperatures of the two, which indicates that when the detection method of the present application uses a probe that is not modified by fluorescent fluorescence for detection, it only needs to further add fluorescent dyes to achieve the purpose of detecting mutant DNA, and can also effectively distinguish wild-type DNA from mutant DNA. At the same time, this embodiment also shows that the detection method of the present application can detect DNA mutations with a mutation frequency of at least 0.2%.

[0110] Example 4

[0111] Detection of EGFR D770delinsGY (c.2308_2309insGTT) site variation

[0112] Step 1: Design primers and probes:

[0113] Upstream primer: 5'-CTACGTGATGGCCAGC-3' (SEQ ID NO: 10)

[0114] Downstream primer: 5'-GGCATGAGCTGCGTGATGAG-3' (SEQ ID NO: 11)

[0115] Probe: VIC-5'- ctga G CCAGC GTGG-ACAACC Ctcag -3'-BHQ1 (the middle sequence is SEQ ID NO: 12)

[0116] Among them, the single underline mark of the probe refers to the complementary sequence of the hairpin structure, the italic mark refers to the overlapping part of the probe and the upstream primer sequence, and the "-" in the middle position of the probe sequence refers to the mutant DNA insertion variant base site in this embodiment, and the inserted variant base is GTT.

[0117] Step 2: The PCR reaction system is prepared as shown in Table 7.

[0118] Table 7:

[0119]

[0120] Step 3: Referring to the reaction system in step 2, use 0.2%, 0.5%, 1%, mutant frequency DNA and wild-type DNA as DNA templates to prepare one reaction each, and perform the reaction in a Macrostone 96S qPCR instrument according to the reaction program in Table 8 below.

[0121] Table 8:

[0122]

[0123] Step 4: Observation of results: See the test results Figure 5 .

[0124] from Figure 5 It can be seen from the test results that no melting peak was found between the PCR amplification product of wild-type DNA and the probe, which indicates that during the annealing process of PCR amplification, the wild-type DNA was completely bound by the probe, resulting in the inability of the wild-type DNA to bind to the primer, resulting in the complete suppression of the amplification of the wild-type DNA, while the PCR amplification product of the mutant DNA and the probe formed a melting peak at about 56°C, which indicates that in the detection method of the present application, multi-site mutations are easier to detect than single-site mutations. The applicant analyzed that this is because after multi-site mutations occur, the difference in Tm between the probe and the wild-type DNA and the mutant DNA will be greater, which can better suppress the amplification of the wild-type DNA, or even completely suppress the amplification of the wild-type DNA. At the same time, this embodiment also shows that the detection method of the present application can detect DNA mutations with a mutation frequency of at least 0.2%-1%.

[0125] Example 5

[0126] Detection of 10 bp deletion mutation at microsatellite locus NR27

[0127] Step 1: Design primers and probes:

[0128] Upstream primer: 5'-CAAACCACTGGTAAAAAAAA-3' (SEQ ID NO: 13)

[0129] Downstream primer: 5'-CGATAATACTAGCAATGACCA-3' (SEQ ID NO: 14)

[0130] Probe:

[0131] VIC-5'- CGA GGTA AAAAAAA AAAAAAAAAAAAAAAAAAG TACCTCG -3'-BHQ1 (the middle sequence is SEQ ID NO: 15)

[0132] The single underline mark of the probe refers to the complementary sequence of the hairpin structure, and the italic mark refers to the overlapping part of the probe and the upstream primer sequence.

[0133] The mutant DNA in this example lacks 10 bp.

[0134] Step 2: The PCR reaction system is prepared as shown in Table 9.

[0135] Table 9:

[0136]

[0137] Step 3: Referring to the reaction system in step 2, use 0.5%, 1%, 2% mutation frequency DNA and wild-type DNA as DNA templates to prepare one reaction each, and perform the reaction in a Macrostone 96S qPCR instrument according to the reaction program in Table 10 below.

[0138] Table 10:

[0139]

[0140] Step 4: Observation of results: See the test results Figure 6 .

[0141] from Figure 6 It can be seen from the test results that the melting temperature of the melting peak formed by the PCR amplification product of wild-type DNA and the probe is around 55°C, while for the mutant DNA with a mutation frequency of 2%, 0.5%, and 1% of NR270 with a deletion of 10bp, in addition to the melting peak at around 55°C, there is also a melting peak formed by the PCR amplification product of mutant DNA and the probe at a lower temperature of around 40°C, which indicates that the detection method of the present application can achieve the purpose of detecting variant DNA through the feature of inconsistent melting peaks, and can effectively distinguish wild-type DNA from mutant DNA. At the same time, this embodiment also shows that the detection method of the present application can detect DNA mutations with a mutation frequency of at least 0.5%-1%.

[0142] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A probe and primer for detecting nucleic acid variation based on a melting curve method, characterized in that: The primers are composed of an upstream primer and a downstream primer, the Tm value of the upstream primer is less than the Tm value of the probe; the probe is a hairpin structure probe, and from the 5' end to the 3' end, there are a stem, a loop and a stem in sequence, the length of the stem is 4-8 bp, the two stems are complementary sequences, the length of the loop is 13-40 bp, and the sequence of the loop is the same as or complementary to the sequence of the wild-type DNA; the 3' end of the probe has a modification that can block polymerase extension, and the partial sequence of the probe close to the 5' end overlaps with the partial sequence starting from the 3' end of the upstream primer; The probes are shown in SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, and SEQ ID NO:15, the upstream primers are shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:13, and the downstream primers are shown in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, and SEQ ID NO:

14.

2. A probe and primer for detecting nucleic acid variation based on melting curve method according to claim 1, characterized in that: Modifications used at the 3' end of the probe that can block polymerase extension include 3'ddC, 3'InverteddT, 3'C3spacer, 3'amino, 3'phosphorylation, and quencher groups.

3. A probe and primer for detecting nucleic acid variation based on melting curve method according to claim 2, characterized in that: The quenching groups include BHQ1, BHQ2, BHQ3, TAMARA and Dabcyl.

4. The probe and primer for detecting nucleic acid variation based on melting curve method according to claim 1, characterized in that: The probe is a fluorescent modified probe, and a fluorescent group is connected to the 5' end of the fluorescent modified probe.

5. A probe and primer for detecting nucleic acid variation based on melting curve method according to claim 4, characterized in that: The fluorescent groups include FAM, TET, JOE, VIC, HEX, NED, ROX, Tamara, Cy3 and Cy5.

6. Use of the probe and primer for detecting nucleic acid variation based on the melting curve method as described in any one of claims 1 to 5 in the preparation of a detection kit for detecting KRAS G12C site variation, KRAS A146T site variation, KRAS A146V site variation, EGFR D770delinsGY site variation, and microsatellite site NR27 deletion 10bp variation based on the melting curve method.

7. The use according to claim 6, characterized in that: The following steps are involved: Step 1, design probes and primers; Step 2, configuration of PCR reaction system: such as formula 1 or formula 2; The formula 1 consists of a buffer, dNTPs, an upstream primer, a downstream primer, a fluorescent modified probe, a DNA polymerase, a DNA template and nuclease-free water; Formula 2 consists of a buffer, dNTPs, an upstream primer, a downstream primer, a probe without fluorescence modification, a fluorescent dye, a DNA polymerase, a DNA template, and nuclease-free water; Step 3, PCR amplification reaction; The PCR amplification reaction procedure comprises: the first step: pre-denaturation, 90-95°C, 7-8min, 1 cycle in total; the second step: amplification, 90-95°C, 10-60s, 50-72°C, 30-120s, 40-60 cycles in total; the third step: denaturation, 90-95°C, 2-3min, 1 cycle in total; the fourth step: renaturation, 25-45°C, 1-2min, 1 cycle in total; Step 4, melting curve detection: heating range 25-90°C, heating rate 0.02-0.4°C / s, a total of 1 cycle; Step 5: Analysis of melting curve detection results.

8. The use according to claim 7, characterized in that: The fluorescent dyes include SYBR green I, EvaGreen and LCgreen.

Images