BRAF, KRAS and TERT gene mutation digital PCR detection reagent and application

Abstract

The invention provides a BRAF, KRAS and TERT gene mutation digital PCR detection reagent and application thereof. Specifically, the invention provides an optimized primer pair, an amplification method, a nucleic acid probe and a detection system aiming at sequences where BRAF, KRAS and TERT gene mutations are located, and further provides a kit for detecting the BRAF, KRAS and TERT gene mutations. According to the invention, the BRAF, KRAS and TERT gene mutation can be detected with high sensitivity and strong specificity for different samples by optimizing the primer pair, the probe and corresponding reaction conditions.

Description

Technical Field

[0001] The present invention relates to the field of gene detection, and in particular, to a digital PCR detection reagent and application for detecting BRAF, KRAS, and TERT gene mutations. Background Art

[0002] In recent years, the incidence of thyroid cancer has shown a significant upward trend. Although the overall prognosis of differentiated thyroid cancer (DTC) is relatively good, some cases still present clinical challenges such as invasive growth, metastasis, and radioiodine resistance. Research has shown that the synergistic effects of BRAF V600E, KRAS, and TERT promoter gene mutations are closely related to the occurrence, development, pathological classification, and treatment resistance of thyroid cancer. The BRAF V600E mutation, as a core molecular marker of papillary thyroid cancer (PTC), is significantly associated with increased risks of tumor invasion, lymph node metastasis, and postoperative recurrence; the KRAS mutation shows specific activation characteristics in follicular thyroid cancer (FTC) and poorly differentiated cancer, suggesting its possible involvement in the abnormal regulation of the MAPK / ERK signaling pathway; while the TERT promoter mutation has been confirmed as an independent predictor of thyroid cancer dedifferentiation and poor prognosis, showing a strong correlation with shortened overall survival of patients.

[0003] Currently, the clinical detection system mostly adopts a single-gene step-by-step detection strategy, which has technical bottlenecks such as a long detection cycle (>72 hours), high sample consumption (requiring ≥3 consecutive sections), and insufficient detection sensitivity (the detection lower limit of the conventional PCR method is only 1-5%). Especially for thyroid nodules with uncertain fine needle aspiration cytology (FNA) diagnosis, the existing technology is difficult to synchronously obtain multi-dimensional molecular typing information from trace samples, resulting in the inability to achieve accurate risk stratification in about 30% of cases. In addition, although next-generation sequencing (NGS) platforms can achieve multi-gene detection, their operational complexity, high cost, and data analysis delay seriously limit their clinical popularization and application.

[0004] Therefore, it is of great significance in this field to achieve the synchronous detection of BRAF V600E (c.1799T>A), KRAS (hotspot regions of G12 / G13 codons), and TERT (C228T / C250T) mutations in thyroid tissue / cytology samples. Summary of the Invention

[0005] The object of the present invention is to provide a method for detecting target targets quickly, with high specificity, high sensitivity, and strong anti-interference ability, so as to solve the problems of long time and low sensitivity of existing detection technologies.

[0006] The object of the present invention is to provide a detection method and reagent for synchronously detecting BRAF V600E (c.1799T>A), KRAS (hotspot regions of G12 / G13 codons), and TERT (C228T / C250T) mutations in thyroid tissue / cytology samples with high sensitivity and high specificity.

[0007] In the first aspect of the present invention, a reagent for detecting gene mutations is provided, and the reagent includes: (a) A first primer pair for detecting BRAF V600E mutation, the first primer pair includes the primers shown in SEQ ID NO: 1 and 2; and a first probe for detecting BRAF V600E mutation used in combination with the first primer pair, the first probe is a combination of the following probes: the probe shown in SEQ ID NO: 7, the probe shown in SEQ ID NO: 8; and (b) A second primer pair for detecting mutations in the KRAS G12 and G13 hotspot regions, the second primer pair includes the primers shown in SEQ ID NO: 21 and 22; and a second probe for detecting mutations in the KRAS G12 and G13 hotspot regions used in combination with the second primer pair, the second probe is a combination of the following probes: the probe shown in SEQ ID NO: 31, the probe shown in SEQ ID NO: 34, the probe shown in SEQ ID NO: 36, the probe shown in SEQ ID NO: 39.

[0008] In another preferred example, the reagent further includes: (c) A third primer pair for detecting TERT promoter C228T mutation and C250T mutation, the third primer pair includes the primers shown in SEQ ID NO: 9 and 10; and a third probe for detecting TERT promoter C228T mutation and C250T mutation used in combination with the third primer pair, the third probe is a combination of the following probes: the probe shown in SEQ ID NO: 19, the probe shown in SEQ ID NO: 20.

[0009] In another preferred example, the BRAF V600E mutation refers to the mutation of valine V at position 600 of the BRAF protein amino acid sequence to glutamic acid E (i.e., V600E).

[0010] In another preferred example, the KRAS G12 and G13 hotspot region mutations include the following mutations: (1) The mutation of glycine G at position 12 of the KRAS protein amino acid sequence to alanine A (i.e., G12A); (2) The glycine G at position 12 of the KRAS protein amino acid sequence is mutated to cysteine C (i.e., G12C); (3) The glycine G at position 12 of the KRAS protein amino acid sequence is mutated to aspartic acid D (i.e., G12D); (4) The glycine G at position 12 of the KRAS protein amino acid sequence is mutated to serine S (i.e., G12S); (5) The glycine G at position 12 of the KRAS protein amino acid sequence is mutated to arginine R (i.e., G12R); (6) The glycine G at position 12 of the KRAS protein amino acid sequence is mutated to valine V (i.e., G12V); (7) The glycine G at position 13 of the KRAS protein amino acid sequence is mutated to cysteine C (i.e., G13C); (8) The glycine G at position 13 of the KRAS protein amino acid sequence is mutated to aspartic acid D (i.e., G13D).

[0011] In another preferred example, the TERT promoter C228T mutation refers to the cytosine C at position 228 of the TERT promoter gene nucleotide sequence being mutated to thymine T (TERT c.228 C>T); the TERT promoter C250T mutation refers to the cytosine C at position 250 of the TERT promoter gene nucleotide sequence being mutated to thymine T (TERT c.250 C>T).

[0012] In another preferred example, the sequence of SEQ ID NO: 7 is: CGAGATTTC+T+CTGTAGCT; the sequence of SEQ ID NO: 8 is: TCGAGATTT+C+ACTGTAGC; In each formula, "+T" represents locked nucleic acid T, "+C" represents locked nucleic acid C, and "+A" represents locked nucleic acid A.

[0013] In another preferred example, the sequence of SEQ ID NO: 31 is: AGCT+G+GT+G+GCG; the sequence of SEQ ID NO: 34 is: AGCT+G+a+TGGCGTAgg; the sequence of SEQ ID NO: 36 is: tggAGC+T+A+GTGGCGT; the sequence of SEQ ID NO: 39 is: AGCTGG+TG+AC+GTAgg; In each formula, "+T" represents locked nucleic acid T, "+G" represents locked nucleic acid G, and "+A" represents locked nucleic acid A.

[0014] In another preferred example, the sequence of SEQ ID NO: 19 is: ACCCC+T+C+CCGGG; the sequence of SEQ ID NO: 20 is: AGCCC+C+C+TCCGG; In each formula, "+T" represents locked nucleic acid T, and "+C" represents locked nucleic acid C.

[0015] In another preferred example, the structure of the first probe 5'-3' is shown in formula (I): Z1-Z2-Z3 (I) Wherein, Z1 is a fluorescent group; Z2 is a specific complementary nucleic acid sequence containing or not containing locked nucleic acids; Z3 is a quenching group; "-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.

[0016] In another preferred example, the specific nucleic acid sequence of Z2 targets the wild-type BRAF V600 site.

[0017] In another preferred example, the specific nucleic acid sequence of Z2 targets the mutant BRAF V600E site.

[0018] In another preferred example, Z2 contains locked nucleic acid modifications.

[0019] In another preferred example, the modification sites of the locked nucleic acid modification cover the mutant base and 2-4 nucleotides upstream and downstream thereof.

[0020] In another preferred example, the sequence of Z2 is selected from the following group: The sequence shown in SEQ ID NO: 7: CGAGATTTC+T+CTGTAGCT; The sequence shown in SEQ ID NO: 8: TCGAGATTT+C+ACTGTAGC; In each formula, "+T" represents locked nucleic acid T, "+C" represents locked nucleic acid C, and "+A" represents locked nucleic acid A.

[0021] In another preferred example, the fluorescent groups are independently located at the 5'-end, 3'-end, and middle of the nucleic acid probe.

[0022] In another preferred example, the fluorescent group and the quenching group are independently located at the 5'-end, 3'-end, and / or middle.

[0023] In another preferred example, the fluorescent group includes a fluorescent group crosslinked to the DNA probe.

[0024] In another preferred example, the fluorescent group is selected from the group consisting of: FAM, HEX, ROX, VIC, FITC, BODIPY-FL, G-Dye100, FluorX, Cy3, Cy5, Cy5.5, Texas Red, or a combination thereof.

[0025] In another preferred example, the quenching group is selected from the group consisting of: DABCYL, TAMRA, BHQ 1, BHQ 2, BHQ3, MGB, BBQ-650, TQ1-TQ6, QSY 7 carboxylic acid, TQ7, eclipse, or a combination thereof.

[0026] In another preferred example, the structure of the second probe from 5'-3' is shown as formula (I): Z1’-Z2’-Z3’ (II) Wherein, Z1’ is a fluorescent group; Z2’ is a specific complementary nucleic acid sequence containing or not containing locked nucleic acid; Z3’ is a quenching group; "-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.

[0027] In another preferred example, the 3' end of the probe shown in SEQ ID NO: 31 is subjected to a blocking treatment.

[0028] In another preferred example, the 3' end blocking treatment method includes: 3' end phosphorylation, C3 spacer.

[0029] In another preferred example, the Z2' specific nucleic acid sequence targets the wild-type KRAS G12 site and / or the wild-type KRAS G13 site.

[0030] In another preferred example, the Z2' specific nucleic acid sequence targets a site selected from the group consisting of: (1) mutant KRAS G12A site; (2) mutant KRAS G12C site; (3) mutant KRAS G12D site; (4) mutant KRAS G12S site; (5) mutant KRAS G12R site; (6) mutant KRAS G12V site; (7) mutant KRAS G13C site; (8) mutant KRAS G12D site; or a combination thereof.

[0031] In another preferred example, the Z2' contains locked nucleic acid modification.

[0032] In another preferred embodiment, the modified sites of the locked nucleic acid modification cover the mutated base and 2-4 nucleotides upstream and downstream thereof.

[0033] In another preferred embodiment, the sequence of the said Z2 is selected from the following group: The sequence shown in SEQ ID NO: 31: AGCT+G+GT+G+GCG; The sequence shown in SEQ ID NO: 34: AGCT+G+a+TGGCGTAgg; The sequence shown in SEQ ID NO: 36: tggAGC+T+A+GTGGCGT; The sequence shown in SEQ ID NO: 39: AGCTGG+TG+AC+GTAgg; In each formula, "+T" represents locked nucleic acid T, "+G" represents locked nucleic acid G, and "+A" represents locked nucleic acid A.

[0034] In another preferred embodiment, the structure of the third probe from 5'-3' is shown as formula (I): Z1''-Z2''-Z3'' (III) Wherein, Z1'' is a fluorescent group; Z2'' is a specific complementary nucleic acid sequence containing or not containing locked nucleic acid; Z3'' is a quenching group; "-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.

[0035] In another preferred embodiment, the specific nucleic acid sequence of the said Z2'' targets the wild-type TERT C228 site and / or the wild-type TERT C250 site.

[0036] In another preferred embodiment, the specific nucleic acid sequence of the said Z2'' targets the mutant TERT C228T site and / or the wild-type TERT C250T site.

[0037] In another preferred embodiment, the said Z2'' contains locked nucleic acid modification.

[0038] In another preferred embodiment, the modified sites of the locked nucleic acid modification cover the mutated base and 2-4 nucleotides upstream and downstream thereof.

[0039] In another preferred embodiment, the sequence of the said Z2'' is selected from the following group: The sequence shown in SEQ ID NO: 19: ACCCC+T+C+CCGGG; The sequence shown in SEQ ID NO: 20: AGCCC+C+C+TCCGG; In each formula, "+T" represents locked nucleic acid T, and "+C" represents locked nucleic acid C.

[0040] In a second aspect of the present invention, a kit is provided, which contains the reagent for detecting gene mutation described in the first aspect of the present invention.

[0041] In another preferred example, the primers and probes in the reagent exist in the form of a primer-probe mixture.

[0042] In another preferred example, the kit further includes a positive control product and a negative control product.

[0043] In another preferred example, the positive control product is: a mixture of BRAF V600E mutant cell line RKO genomic DNA and wild-type cell line 293T genomic DNA (mutation rate 3%), a mixture of TERT promoter C228T mutant cell line HepG genomic DNA and wild-type cell line 293T genomic DNA (mutation rate 1%), a mixture of artificially synthesized TERT promoter C250T mutant plasmid and wild-type cell line 293T genomic DNA (mutation rate 1%), a mixture of KRAS G12 / G13 hotspot mutant cell lines Sw1116, A549, CAL-62, HCT-116, NCL-H1734 genomic DNA and artificially synthesized plasmids and wild-type cell line 293T genomic DNA respectively (mutation rate 1-3%); the concentration is about 3000 copies / ul.

[0044] In another preferred example, the negative control product is: wild-type cell line 293T genomic DNA (concentration about 3000 copies / ul).

[0045] In a third aspect of the present invention, a method for non-diagnostically detecting in vitro whether a test sample contains a gene mutation is provided, including the steps: (s1) Provide a PCR reaction system, which contains the test sample as a template and the reagent for detecting gene mutation described in the first aspect of the present invention; (s2) Perform a PCR reaction on the PCR reaction system in step (S1) to obtain an amplification product; (s3) Analyze the amplification product generated in step (S2) to obtain an analysis result of whether the test sample contains a gene mutation.

[0046] In another preferred example, the reagent further includes: (c) A third primer pair for detecting TERT promoter C228T mutation and C250T mutation, the third primer pair comprising the primers shown in SEQ ID NO: 9 and 10; and a third probe for detecting TERT promoter C228T mutation and C250T mutation used in combination with the third primer pair, the third probe being a combination of the following probes: the probe shown in SEQ ID NO: 19, the probe shown in SEQ ID NO: 20.

[0047] In another preferred example, the sample to be tested is selected from the group consisting of: formalin-fixed paraffin-embedded (FFPE) tissue section samples, fine needle aspiration (FNA) cytology samples, or combinations thereof.

[0048] In another preferred example, the required amount of the sample is that the thickness of a single section is ≤5 μm or the number of cells is ≥100.

[0049] In another preferred example, in step (s1), the reaction system further contains: 5-20% glycerol and 0.01-1% Poloxamer 188.

[0050] In another preferred example, in step (s1), the reaction system further contains: 10% glycerol and 0.5% Poloxamer 188.

[0051] In another preferred example, in the reaction system, the probe for detecting BRAF V600E mutation includes a wild-type probe for detecting the BRAF V600 site and a mutant probe for detecting the BRAF V600E site; and the fluorescence reporter group of the wild-type probe is HEX, and the fluorescence reporter group of the mutant probe is FAM.

[0052] In another preferred example, in the reaction system, the probe for detecting KRAS G12 and G13 hotspot region mutations includes a wild-type probe for detecting the KRAS G12 and G13 sites and a mutant probe for detecting the mutant sites of the KRAS G12 and G13 hotspot region mutations; the 5' end of the wild-type probe has no fluorescence reporter group, and the 3' end is phosphorylated and blocked; the fluorescence reporter groups of the mutant probes are all Cy5.5.

[0053] In another preferred example, the probe for detecting TERT promoter C228T mutation and C250T mutation includes a first wild-type probe for detecting the TERT promoter C228 site and a second wild-type probe for detecting the TERT promoter C250 site; the fluorescence reporter group of the first wild-type is Cy5, and the fluorescence reporter group of the second wild-type probe is ROX.

[0054] In another preferred example, the analysis result is a qualitative result.

[0055] In another preferred example, the PCR reaction system is a digital PCR reaction system.

[0056] In another preferred example, among the reagents, the probes for detecting BRAF and KRAS mutant genes adopt different fluorescent reporter groups.

[0057] In another preferred example, the method is non-diagnostic and non-therapeutic.

[0058] In another preferred example, the method is an in vitro method.

[0059] In another preferred example, the detection sensitivity of the method is 0.1% mutant allele frequency (MAF).

[0060] In another preferred example, the lower limit of detection of the method is 5 copies / reaction system of mutant DNA.

[0061] In another preferred example, the detection period of the method is ≤ 2 hours.

[0062] In the fourth aspect of the present invention, there is provided the use of the reagent for detecting mutations as described in the first aspect of the present invention or the kit as described in the second aspect of the present invention for preparing a diagnostic product, and the diagnostic product is used for: (a) Predicting tumor invasiveness and the risk of postoperative recurrence (high-risk determination criterion: the simultaneous presence of ≥ 2 gene mutations); (b) Screening cases sensitive to targeted therapy with lenvatinib and sorafenib (mutation abundance threshold ≥ 5%).

[0063] In another preferred example, when ≥ 2 gene mutations are present simultaneously, it is determined that the object to be tested has a high risk of tumor invasion or postoperative recurrence.

[0064] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Shows the amplification effect of 3 pairs of primers for specifically amplifying the BRAF V600E mutation site in Example 1 (PCR electrophoresis result of primer screening).

[0066] Figure 2 Shows the digital PCR two-dimensional map of primer P1-F / R combined with a probe for detecting BRAF V600E in Example 1.

[0067] Figure 3 Shows the digital PCR two-dimensional map of detecting BRAF V600E with primer P2-F / R and probe in Example 1.

[0068] Figure 4 Shows the digital PCR two-dimensional map of detecting BRAF V600E with primer P3-F / R and probe in Example 1.

[0069] Figure 5 Shows the amplification effect of 5 pairs of primers used for specifically amplifying the mutation position of the TERT promoter in Example 2.

[0070] Figure 6 Shows the digital PCR two-dimensional map of detecting the wild-type TERT promoter with the selected primer pair and probe in Example 2.

[0071] Figure 7 Shows the digital PCR two-dimensional map of detecting the wild-type TERT promoter after optimizing the reaction system in Example 3.

[0072] Figure 8 Shows the digital PCR two-dimensional map of detecting the TERT promoter C228T mutation after optimizing the reaction system in Example 3.

[0073] Figure 9 Shows the digital PCR two-dimensional map of detecting the TERT promoter C250T mutation after optimizing the reaction system in Example 3.

[0074] Figure 10 Shows the amplification effect of 5 pairs of primers used for specifically amplifying the hot spot region of KRAS G12 / G13 in Example 4.

[0075] Figure 11 Shows the digital PCR two-dimensional map of detecting the KRAS G12A mutation in Example 5.

[0076] Figure 12 Shows the digital PCR two-dimensional map of detecting the KRAS G12C mutation in Example 5.

[0077] Figure 13 Shows the digital PCR two-dimensional map of detecting the KRAS G12D mutation in Example 5.

[0078] Figure 14 Shows the digital PCR two-dimensional map of detecting the KRAS G12S mutation in Example 5.

[0079] Figure 15 Shows the digital PCR two-dimensional map of detecting the KRAS G12R mutation in Example 5.

[0080] Figure 16 Shows the digital PCR two-dimensional map for detecting KRAS G12V mutation in Example 5.

[0081] Figure 17 Shows the digital PCR two-dimensional map for detecting KRAS G13C mutation in Example 5.

[0082] Figure 18 Shows the digital PCR two-dimensional map for detecting KRAS G13D mutation in Example 5.

[0083] Figure 19 Shows the digital PCR two-dimensional map for detecting KRAS G12A mutation in Example 6.

[0084] Figure 20 Shows the digital PCR two-dimensional map for detecting KRAS G12C mutation in Example 6.

[0085] Figure 21 Shows the digital PCR two-dimensional map for detecting KRAS G12D mutation in Example 6.

[0086] Figure 22 Shows the digital PCR two-dimensional map for detecting KRAS G12S mutation in Example 6.

[0087] Figure 23 Shows the digital PCR two-dimensional map for detecting KRAS G12R mutation in Example 6.

[0088] Figure 24 Shows the digital PCR two-dimensional map for detecting KRAS G12V mutation in Example 6.

[0089] Figure 25 Shows the digital PCR two-dimensional map for detecting KRAS G13C mutation in Example 6.

[0090] Figure 26 Shows the digital PCR two-dimensional map for detecting KRAS G13D mutation in Example 6. Detailed implementation manners

[0091] Through extensive and in-depth research, the present inventors, through a large number of screenings, especially by optimizing primer sequences and probe sequences, and combined with a digital PCR platform, can effectively improve the detection effect of gene mutations, and break through the technical bottlenecks such as long detection cycle, high sample consumption, and insufficient detection sensitivity in the existing gene mutation detection technologies. A method with high specificity, high sensitivity, and strong anti-interference ability for simultaneously detecting BRAF, KRAS, and TERT gene mutations is provided. On this basis, the present inventors completed the present invention.

[0092] Specifically, the present invention provides a method for detecting BRAF V600E (c.1799T>A), KRAS (G12A, G12C, G12D, G12S, G12R, G12V, G13VC and G13D), and TERT promoter C228T and TERT promoter C250T mutant genes. By designing and optimizing primer and probe sequences, a primer and probe composition that can be used to simultaneously detect the above three gene mutations without interference with each other in the same reaction was finally screened, and a digital PCR detection system was established to qualitatively and quantitatively detect the mutation status of the above genes. When the method and reagent of the present invention are used to detect the above gene mutations, they have unexpectedly high sensitivity and high specificity, and can detect samples with different difficulties.

[0093] Term To facilitate a better understanding of the present disclosure, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0094] The term "about" can refer to a value or a component within an acceptable error range of a particular value or component determined by a person of ordinary skill in the art, which will depend in part on how the value or component is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0095] As used herein, the term "comprising" or "including" can be open-ended, semi-closed, and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0096] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window, which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein, and determining the number of positions where identical residues occur. Generally, this is expressed as a percentage. Methods for measuring sequence identity of nucleotide sequences are well known to those skilled in the art.

[0097] Digital PCR (digital PCR) technology Digital PCR technology is an absolute quantitative method for nucleic acid quantification based on the single-molecule PCR method. It mainly uses the microfluidics or droplet method in the current hot research field of analytical chemistry to disperse a large amount of diluted nucleic acid solution into the microreactor or droplets of the chip, and the number of nucleic acid templates in each reactor is less than or equal to 1. After the PCR cycle, the fluorescence signal of each droplet is analyzed after the amplification is completed. The reactor with the nucleic acid molecule template will give a fluorescent signal, and the reactor without the template will not have a fluorescent signal. According to the relative proportion and the volume of the reactor, the nucleic acid concentration of the original solution can be calculated.

[0098] Compared with conventional qPCR, digital PCR can accurately quantify and detect target nucleic acid molecules with high sensitivity. The method for analyzing the results of conventional qPCR is an analog method, wherein the digital PCR method, the results of which are analyzed by a digital method (because the obtained signal has a value of "0" or "1"), has the advantages of being able to analyze large volume samples, simultaneously detect different samples, and simultaneously perform different tests. Digital PCR technology is a technology that can use a single molecule counting method without a standard curve to absolutely quantify DNA samples, and can perform more accurate absolute quantification of a single droplet per well by PCR (see Gudrun Pohl and le-Ming Shih, Principle and applications of digital PCR, Expert Rev. Mol. Diagn. 4 (1), 41-47 (2004)). Digital PCR has the advantages of high sensitivity, accurate quantification without a standard curve, and simple operation.

[0099] In digital PCR, each droplet containing a sample gene template prepared so as to be diluted to an average copy number of 0.5-1, amplification primers, and a fluorescent probe is dispensed into a single well, and microemulsion PCR is performed. Then, the wells showing a fluorescent signal are counted as a value of "1" because a sample with a gene copy number of 1 is dispensed into the well and shows a fluorescent signal after amplification, while the wells showing no signal are counted as "0" because a sample with a gene copy number of 0 is dispensed into the well and does not show a fluorescent signal due to no amplification. In this way, absolute quantification can be achieved.

[0100] Primers Primer refers to a macromolecule with a specific nucleotide sequence that is stimulated to synthesize at the beginning of nucleotide polymerization and is covalently linked to the reactant. Primers are usually two artificially synthesized oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the target region, and the other primer is complementary to another DNA template strand at the other end of the target region.

[0101] In the present invention, in order to improve the sensitivity of the detection system, the corresponding gene fragments in the detection system are pre-amplified, and thus primers corresponding to the sequence where the mutation is located are designed.

[0102] In a preferred example, for the sequence where BRAF V600E is located, multiple pairs of primers are designed upstream and downstream of the V600 site of the BRAF gene. After experimental testing, the primer pair is finally determined to be SEQ ID NO: 1 and 2; for the sequences where TERT promoter C228T and C250T are located, multiple pairs of primers are designed upstream and downstream of the C228 and C250 sites of the TERT gene promoter respectively. After testing, the optimal primer pair is finally determined to be SEQ ID NO: 9 and 10; for the sequence where the KRAS G12 / G13 hotspot region is located, multiple pairs of primers are designed upstream and downstream of the G12 and G13 sites of the KRAS gene respectively. After experimental testing, the optimal primer pair is finally determined to be SEQ ID NO: 21 and 22.

[0103] Probe In this article, "probe", "nucleic acid probe", and "gene probe" can be replaced, and refer to a nucleic acid sequence (DNA or RNA) with a detection label and a known sequence that is complementary to the target gene. The gene probe binds to the target gene through molecular hybridization, generating a hybridization signal, and can display the target gene from the vast genome. According to the hybridization principle, the nucleic acid sequence as a probe must at least meet the following two conditions: ① It should be single-stranded. If it is double-stranded, it must be denatured first; ② It should carry a label that is easily detectable. The nucleic acid probe can include the entire gene or only a part of the gene; it can be DNA itself or RNA transcribed therefrom. In the present invention, the probe also refers to a modified primer with chemical modification groups at both ends or in the middle, and these chemical modifications have special functions including but not limited to: signal indication, enhancing the connection with reactants, etc.

[0104] In another preferred example, the structure of the first probe is shown as formula (I) from 5'-3': Z1-Z2-Z3 (I) Wherein, Z1 is a fluorescent group; Z2 is a specific complementary nucleic acid sequence containing or not containing locked nucleic acid; Z3 is a quenching group; "-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.

[0105] In another preferred example, the specific nucleic acid sequence of Z2 targets the wild-type BRAF V600 site.

[0106] In another preferred embodiment, the Z2 specific nucleic acid sequence targets the mutant BRAF V600E site.

[0107] In another preferred embodiment, the Z2 contains locked nucleic acid modifications.

[0108] In another preferred embodiment, the sequence of the Z2 is selected from the group consisting of: The sequence shown in SEQ ID NO: 7: CGAGATTTC+T+CTGTAGCT; The sequence shown in SEQ ID NO: 8: TCGAGATTT+C+ACTGTAGC; In each formula, "+T" represents locked nucleic acid T, "+C" represents locked nucleic acid C, and "+A" represents locked nucleic acid A.

[0109] In another preferred embodiment, the fluorescent groups are independently located at the 5'-end, 3'-end, and middle of the nucleic acid probe.

[0110] In another preferred embodiment, the fluorescent group and the quenching group are independently located at the 5'-end, 3'-end, and / or middle.

[0111] In another preferred embodiment, the fluorescent group includes a fluorescent group crosslinked to the DNA probe.

[0112] In another preferred embodiment, the fluorescent group is selected from the group consisting of: FAM, HEX, ROX, VIC, FITC, BODIPY-FL, G-Dye100, FluorX, Cy3, Cy5, Cy5.5, Texas Red, or a combination thereof.

[0113] In another preferred embodiment, the quenching group is selected from the group consisting of: DABCYL, TAMRA, BHQ 1, BHQ 2, BHQ3, MGB, BBQ-650, TQ1-TQ6, QSY 7 carboxylic acid, TQ7, eclipse, or a combination thereof.

[0114] In another preferred embodiment, the structure of the second probe 5'-3' is as shown in formula (I): Z1'-Z2'-Z3' (II) Wherein, Z1' is a fluorescent group; Z2' is a specific complementary nucleic acid sequence containing or not containing locked nucleic acids; Z3' is a quenching group; "-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.

[0115] In another preferred embodiment, the Z2' specific nucleic acid sequence targets the wild-type KRAS G12 site and / or the wild-type KRAS G13 site.

[0116] In another preferred embodiment, the Z2' specific nucleic acid sequence targets a site selected from the group consisting of: (1) the mutant KRAS G12A site; (2) the mutant KRAS G12C site; (3) the mutant KRAS G12D site; (4) the mutant KRAS G12S site; (5) the mutant KRAS G12R site; (6) the mutant KRAS G12V site; (7) the mutant KRAS G13C site; (8) the mutant KRAS G12D site; or a combination thereof.

[0117] In another preferred embodiment, the Z2' contains locked nucleic acid modifications.

[0118] In another preferred embodiment, the sequence of the Z2 is selected from the group consisting of: the sequence shown in SEQ ID NO: 31: AGCT+G+GT+G+GCG; the sequence shown in SEQ ID NO: 34: AGCT+G+a+TGGCGTAgg; the sequence shown in SEQ ID NO: 36: tggAGC+T+A+GTGGCGT; the sequence shown in SEQ ID NO: 39: AGCTGG+TG+AC+GTAgg; In each formula, "+T" represents locked nucleic acid T, "+G" represents locked nucleic acid G, and "+A" represents locked nucleic acid A.

[0119] In another preferred embodiment, the structure of the third probe 5'-3' is shown in formula (I): Z1''-Z2''-Z3'' (III) Wherein, Z1'' is a fluorescent group; Z2'' is a specific complementary nucleic acid sequence containing or not containing locked nucleic acids; Z3'' is a quenching group; "-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.

[0120] In another preferred embodiment, the Z2'' specific nucleic acid sequence targets the wild-type TERT C228 site and / or the wild-type TERT C250 site.

[0121] In another preferred embodiment, the Z2’’ specific nucleic acid sequence targets the mutant TERT C228T site and / or the wild-type TERT C250T site.

[0122] In another preferred embodiment, the Z2’’ contains locked nucleic acid modifications.

[0123] In another preferred embodiment, the sequence of the Z2’’ is selected from the group consisting of: The sequence shown in SEQ ID NO: 19: ACCCC+T+C+CCGGG; The sequence shown in SEQ ID NO: 20: AGCCC+C+C+TCCGG; In each formula, “+T” represents locked nucleic acid T, and “+C” represents locked nucleic acid C.

[0124] Modifications of primers and probes In the present invention, the nucleic acid sequence of the primer includes an unmodified or modified primer sequence.

[0125] Preferably, the inventors can significantly improve the specificity of the probe by modifying the probe with locked nucleic acid (LNA), thereby improving the sensitivity and specificity of the detection results.

[0126] In a preferred embodiment of the present invention, the modification methods are selected from: Phosphorylation, Biotin, Digoxigenin, internal amino modification, 5'-amino modification, 3'-amino modification, Thiol, Spacer, Phosphorthioate, DeoxyUridine (dU), deoxyInosine (dI), or a combination thereof.

[0127] Phosphorylation modification: 5'-phosphorylation can be used for linkers, cloning and gene construction, and ligase-catalyzed ligation reactions. 3'-phosphorylation can be used in experiments related to resistance to 3'-exonuclease digestion and also to block DNA strand extension reactions catalyzed by DNA polymerase.

[0128] Biotin modification: Biotin labeling of primers can be used for non-radioactive immunoassays to detect proteins, intracellular chemical staining, cell separation, nucleic acid separation, hybridization to detect specific DNA / RNA sequences, ion channel conformational changes, etc.

[0129] Digoxigenin Modification: Digoxigenin is linked to the C5 position of uracil via an 11-atom spacer arm. Hybridized digoxigenin probes can be detected by anti-digoxigenin antibodies. Digoxigenin-labeled probes can be used in various hybridization reactions, such as DNA-DNA hybridization (Southern blotting), DNA-RNA hybridization (Northern blotting), dot blotting, clone hybridization, in situ hybridization, and enzyme-linked immunosorbent assay (ELISA).

[0130] Internal Amino Modification: Internal modification is mainly carried out by adding C6-dT aminolinker to thymine residues. After modification, the amino group is 10 atoms away from the backbone and can be used for further labeling and enzyme linkage (such as alkaline phosphatase). Currently, dT-Dabcyl, dT-Biotin, and dT-Digoxingenin modifications mediated by internal amino modification are provided.

[0131] 5'-Amino Modification: It can be used to prepare functionalized oligonucleotides and is widely used in DNA microarray and multiplex labeling diagnostic systems. Currently, two types of 5' C6 amino modification and 5' C12 amino modification are provided. The former can be used to link some compounds that will not affect their functions even when close to oligonucleotides, and the latter is used for the linkage of affinity purification groups and some fluorescent labels, especially when fluorescence may be quenched due to the label being too close to the DNA strand.

[0132] 3'-Amino Modification: Currently, 3' C6 amino modification is provided. It can be used to design new diagnostic probes and antisense nucleotides. For example, the 5' end can be labeled with highly sensitive 32P or fluorescein while the 3' end can be amino-modified for other linkages. In addition, 3' modification can inhibit exonuclease digestion at the 3' end and thus can be used in antisense experiments.

[0133] Mercapto Modification: 5'-Mercapto is similar to amino modification in many aspects. Mercapto can be used to attach various modifications such as fluorescent labels and biotin. For example, mercapto-linked fluorescent probes can be prepared in the presence of iodoacetic acid and maleimide derivatives. 5'-mercapto modification mainly uses 5'-thiol-modifier monomers (5'-Thiol-Modifier C6-CE Phosphoramidite or Thiol-Modifier C6 S-S CE Phosphoramidite). After modification with 5'-Thiol-Modifier C6-CE monomer, silver nitrate oxidation must be carried out to remove the protecting group (trityl), while after modification with Thiol-Modifier C6 S-S CE monomer, the disulfide bond must be reduced to mercapto with DTT.

[0134] Spacer modification: The Spacer can provide the necessary spacing for oligonucleotide labeling to reduce the interaction between the labeling group and the oligonucleotide, and is mainly applied to the research of DNA hairpin structures and double-stranded structures. The C3 spacer is mainly used to mimic the three-carbon spacing between the 3' and 5' hydroxyl groups of ribose, or to "replace" an unknown base in a sequence. 3'-Spacer C3 is used to introduce a 3' spacer to prevent the action of 3'-exonuclease and 3'-polymerase. Spacer 18 is often used to introduce a strongly hydrophilic group.

[0135] Thiol modification: Thiol-modified oligonucleotides are mainly used to prevent degradation by nucleases in antisense experiments. You can choose full thiolation, but as the number of thiol bases increases, the Tm value of the oligonucleotide will decrease. To reduce this effect, 2-5 bases at both ends of the primer can be thiolated. Usually, 3 bases at the 5' and 3' ends can be selected for thiolation.

[0136] Deoxyuracil modification: Deoxyuracil can be inserted into oligonucleotides to increase the melting temperature of double-strands and thus enhance the stability of double-strands. Replacing each deoxythymidine with deoxyuracil can increase the melting temperature of double-strands by 1.7 °C.

[0137] Deoxyinosine modification: Deoxyinosine is a naturally occurring base. Although it is not a truly universal base, when combined with other bases, it is relatively more stable in terms of mismatching than other bases. The binding ability of deoxyinosine to other bases is dI:dC > dI:dA > dI:dG > dI:dT. Under the catalysis of DNA polymerase, deoxyinosine preferentially binds to dC.

[0138] The main advantages of the present invention include: (a) The present invention uses Taqman probes in combination with digital PCR, which can solve problems such as low sensitivity, poor specificity, high requirements for the type and quality of samples, and complex positive interpretation methods.

[0139] (b) High sensitivity: Since this method uses a digital PCR platform, the reaction system can be divided into approximately 20,000 tiny reactions. In theory, it can detect single-copy mutations and has a sensitivity advantage that cannot be matched by other technologies. The detection method of the present invention can achieve a minimum detection limit of 5 copies / reaction through verification.

[0140] (c) Strong specificity: The designed specific primer probes are respectively targeted at the specific sequences of mutations and can specifically amplify the target positions.

[0141] (d) The detection reagent or detection method of the present invention has loose requirements for the type and quality of samples and strong anti-interference ability. Due to the high sensitivity of the present invention, the applicable sample types include FNA (this sample is relatively easy to obtain, but the sample volume is small).

[0142] (e) The positive interpretation method is simple: Since the present invention uses an absolute quantification method, there is no need to set a control standard curve, nor is it necessary to compare with an internal reference. The result can be determined whether it contains the target mutant template according to the two-dimensional fluorescence map (Table A).

[0143] Table A Detection result table

[0144] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight percentage and weight parts.

[0145] Example 1 Design and screening of BRAF V600E mutation-specific primers and probes 1.1 Materials and methods: (1) Sample source: Positive control: Genomic DNA of BRAF V600E mutant cell line RKO; Negative control: Genomic DNA of BRAF wild-type cell line 293T.

[0146] (2) Primer / probe design: According to the BRAF gene c.1799T>A mutation site and the primer-probe design principle, 3 pairs of candidate primers (primer P1 - primer P3) and 2 modified probes were designed. See Table 1 below. The probes were modified with locked nucleic acid (LNA), covering several bases upstream and downstream of the mutation site. The quenching group was BHQ1, the reporter group of the wild-type WT probe was HEX, and the reporter group of the mutant MT probe was FAM.

[0147] Table 1

[0148] Note: "+A" represents locked nucleic acid A, "+T" represents locked nucleic acid T, and "+C" represents locked nucleic acid C.

[0149] (3) Preliminary screening: Primer screening: Prepare the PCR reaction system: 5×HS Taq Buffer with Mg 2+ 4 μL, dNTPs (10 mM each) 0.5 μL, HotStart Taq DNA Polymerase 0.2 μL, F primer (10 μM) 1 μL, R primer (100 μM) 1 μL, template 2 μL, make up to 20 μL with water.

[0150] Cycling conditions: 95°C for 10 min, 40 cycles (95°C for 30 s, 56°C for 15 s, 72°C for 15 s).

[0151] Detect 10 μL of the PCR product by electrophoresis.

[0152] The amplification effects of three pairs of primers ( Figure 1 ) are all acceptable, and subsequent digital PCR experiments are carried out.

[0153] (4) Digital PCR experiment: Prepare the PCR reaction system: 5×HS Taq Buffer with Mg 2+ 6 μL, dNTPs (10 mM each) 0.75 μL, HotStart Taq DNA Polymerase 0.2 μL, F primer (10 μM) 1 μL, R primer (10 μM) 1 μL, WT probe (10 μM) 0.5 μL, MT probe (10 μM) 0.5 μL, template (wild type or mutant) 2 μL, make up to 30 μL with water.

[0154] Inject the reaction system into the reaction chip on the BioDigital Qing digital PCR instrument according to the instructions. Cycling conditions: 50°C for 10 min, 95°C for 10 min, 40 cycles (95°C for 30 s, 56°C for 15 s, 72°C for 15 s). After the cycling is completed, read the signal on the BioDigital Qing chip analyzer for analysis.

[0155] 1.2 Results The results are as Figures 2 - 4 shown. The primer P1-F / R has the best effect, and this pair of primers is selected to cooperate with the probe for subsequent experiments; the signal of the primer P2-F / R is lower; the primer P3-F / R has double clusters and is excluded.

[0156] Example 2 Design and screening of specific primers and probes for TERT promoter C228T / C250T mutations 2.1 Primer design and screening 2.1.1 Materials and methods (1) Sample source: Positive control: Genomic DNA of the TERT promoter C228T mutant cell line HepG and the artificially synthesized C250T mutant plasmid; Negative control: Genomic DNA of the TERT promoter wild-type cell line 293T.

[0157] (2) Primer design According to the TERT promoter C228T / C250T mutation sites and primer design principles, 5 pairs of candidate primers (P4 - P8) were designed, as shown in Table 2 below.

[0158] Table 2

[0159] (3) Preliminary screening Primer screening: Prepare the PCR reaction system: 5×HS Taq Buffer with Mg 2+ 4 μL, dNTPs (10 mM each) 0.5 μL, HotStart Taq DNA Polymerase 0.2 μL, F primer (10 μM) 1 μL, R primer (100 μM) 1 μL, template 2 μL, add water to make up to 20 μL.

[0160] Cycling conditions: 95°C for 10 min, 40 cycles (95°C for 30 s, 56°C for 15 s, 72°C for 15 s) Detect 10 μL of the PCR product by electrophoresis.

[0161] 2.1.2 Results The results are as Figure 5 shown. Primer P4 - F / R had the correct bands, but there were 2 non-specific bands (the top band), primer P5 - F / R had weak amplification bands and many non-specific bands, the single-band sizes of primer P6 - F / R and primer P7 - F / R were incorrect, and primer P8 - F / R had no obvious amplification bands. Therefore, primer P4 - F / R was selected for subsequent experiments.

[0162] 2.2 Probe design 2.2.1 Design method Based on the fact that C228T and C250T are relatively close within the same amplicon but do not overlap, and the frequency of simultaneous mutations at both sites is very low, two probes targeting the mutant sites but binding to the wild-type template were designed and labeled with different fluorescent groups (see Table 3 below). When the template in the droplet is wild-type, the two probes bind simultaneously and are hydrolyzed, and the droplet has two fluorescent signals at the same time; when the template in the droplet contains a mutation at one of the sites, the probe at this site cannot bind (such as ROX), while the probe at the other site (such as Cy5) binds normally and is hydrolyzed, generating a single fluorescent signal (Cy5).

[0163] Table 3

[0164] Note: "+T" represents locked nucleic acid T, and "+C" represents locked nucleic acid C.

[0165] Digital PCR experiment: Prepare the PCR reaction system: 5×HS Taq Buffer with Mg 2+ 6 μL, dNTPs (10 mM each) 0.75 μL, HotStart Taq DNA Polymerase 0.2 μL, P4-F / R primers (10 μM) 1 μL each, C228T / C250T WT probes (10 μM) 0.5 μL each, template (wild-type or mutant) 2 μL, and make up to 30 μL with water.

[0166] Inject the reaction system into the reaction chip on the BioDigital Blue digital PCR instrument according to the instructions. Cycling conditions: 50°C for 10 min, 95°C for 10 min, 40 cycles (95°C for 30 s, 56°C for 15 s, 72°C for 15 s). After the cycling is completed, read the signals on the BioDigital Blue chip analyzer of the chip for analysis.

[0167] 2.2.2 Results The results are as Figure 6 shown. For the wild-type template, the signal is weak (signal points in the 45° upward diagonal direction), dispersed, and the effect is not good.

[0168] Example 3 Optimization of the reaction system for detecting TERT promoter C228T / C250T mutations Prepare the PCR reaction system according to the system described in Example 2, and additionally add glycerol with a final concentration of 1-20%, or 0.1-1% Poloxamer 188, or 1-10% propylene glycol, or 1-10% ethylene glycol, or 0.5-2M betaine, or a combination thereof. Perform digital PCR detection according to the method in Example 2. It is found that the signal is the best when 10% glycerol and 0.5% Poloxamer 188 are added, see Figures 7 - 9 (in sequence are wild-type template, C228T mutant template (red mutant signal point), and C250T mutant template (yellow mutant signal point)).

[0169] Example 4 Design and Screening of Primers for KRAS G12 / G13 Hotspot Region 4.1 Materials and Methods (1) Sample Source: Positive control: Genomic DNA of KRAS G12 / G13 hotspot mutant cell lines Sw1116, ΜM-UC-3, A549, CAL-62, HCT-116, NCL-H1734 and synthetic plasmids; Negative control: Genomic DNA of KRAS wild-type cell line 293T.

[0170] (2) Primer Design According to the KRAS G12 / G13 hotspot mutation sites and primer-probe design principles, design 5 pairs of candidate primers (P9-P13), as shown in Table 4 below.

[0171] Table 4

[0172] (3) Primer Screening Prepare the ordinary PCR reaction system according to Example 2, use primers P9 to P13, and perform electrophoresis detection after the cycle ends.

[0173] 4.2 Results The results are as Figure 10 shown. All three pairs of primers, P9, P10, and P11, can amplify the correct bands, while the primer pairs P12 and P13 cannot amplify the correct bands. Select primer P9 for subsequent experiments.

[0174] Example 5 Design of Probes for KRAS G12 / G13 Hotspot Mutations According to the primers screened in Example 4 and the probe design principles, design 9 probes, including 1 wild-type WT probe labeled with HEX and BHQ1; the other 8 probes are respectively for 8 mutations, labeled with FAM and BHQ1. As shown in Table 5 below.

[0175] Table 5

[0176] Note: "+A" represents locked nucleic acid A, "+T" represents locked nucleic acid T, "+C" represents locked nucleic acid C, and "+G" represents locked nucleic acid G.

[0177] According to the foregoing examples for digital PCR detection, when the 8 mutant probes are respectively paired with the WT probe, the corresponding mutations can all be detected. The detection results of G12A, G12C, G12D, G12S, G12R, G12V, G13C, and G13D are respectively as Figures 11 - 18 shown.

[0178] Example 6 Detection of 8 mutations in the KRAS G12 / G13 hot spot region using 3 mutant probes For thyroid cancer, the detection of KRAS mutations does not need to distinguish the type of mutation. Therefore, it is considered to use fewer probes to detect 8 mutations. After thermodynamic calculations, four probes, SEQ ID NO.31, 34, 36, and 39 (1 wild-type as an internal reference and 3 mutant probes for detecting mutations), are selected for combination detection.

[0179] The digital PCR detection process is as before. The detection results of G12A, G12C, G12D, G12S, G12R, G12V, G13C, and G13D are respectively as Figures 19 - 26 shown, and this combination of 4 probes can detect 8 mutations.

[0180] Example 7 Simultaneous detection of BRAF V600E, KRAS (G12 / G13 codon hot spot region), and TERT promoter (C228T / C250T) mutations in one reaction To simultaneously detect these mutations, the probes for the KRAS G12 / G13 codon hot spot region, SEQ ID NO. 31, 34, 36, and 39, are resynthesized. Among them, for SEQ ID NO.31 (wild-type probe), the fluorescence channel is removed, and the 3'-end is blocked with phosphorylation (to prevent probe extension, or C3 or C18 or other blocking methods can also be used). The fluorophores of SEQ ID NO. 34, 36, and 39 are changed to Cy5.5, and the quencher group is changed to BHQ3. The BRAF V600E probe and the TERT probe remain the same as those selected in the above examples.

[0181] Prepare the reaction system according to Example 3. Specifically, the reaction system contains 10% glycerol and 0.5% Poloxamer188. Detect their respective positive controls and negative controls.

[0182] The results show that the specificity of the original singleplex digital PCR is still maintained after combination.

[0183] Example 8 Sensitivity of multiplex detection A series of positive samples with low mutation ratios (as shown in Table 6) were prepared and detected according to the primer-probe combination in Example 7. Each sample was detected 20 times repeatedly, and the detection results of each mutation site are summarized in Table 7 below.

[0184] Table 6

[0185] Table 7

[0186] The results showed that when the mutation ratio of each detected mutation site was 0.1%, at least 95% (19 times) positive results could be obtained. Therefore, the minimum detection limit of the detection reagent was determined to be 0.1%.

[0187] Discussion Based on the multiplex digital PCR technology combined with special reaction system additives, the present invention first realized the synchronous detection of BRAF V600E (c.1799T>A), KRAS (hotspot regions of G12 / G13 codons), and TERT (C228T / C250T) mutations in thyroid tissue / cytological samples. By optimizing the primers, probes, and reaction system, the detection sensitivity was increased to 0.1% (able to detect 5 copies / reaction system of mutant DNA), and the detection cycle was shortened to within 2 hours. Compared with the prior art, while maintaining the compatibility with paraffin-embedded samples, the sample requirement of this kit was significantly reduced to a single section (5 μm thick), which was especially suitable for rapid molecular diagnosis of preoperative FNA specimens.

[0188] The present invention overcomes the technical defects of insufficient sensitivity of traditional PCR and complex operation of NGS, and provides an efficient and low-cost solution for the precise diagnosis and treatment of thyroid cancer.

[0189] All documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A reagent for detecting gene mutations, characterized in that, The reagent includes: (a) A first primer pair for detecting BRAF V600E mutation, the first primer pair including the primers shown in SEQ ID NO: 1 and 2; and a first probe for detecting BRAF V600E mutation used in combination with the first primer pair, the first probe being a combination of the following probes: the probe shown in SEQ ID NO: 7, the probe shown in SEQ ID NO: 8; and (b) A second primer pair for detecting mutations in the hot regions of KRAS G12 and G13, the second primer pair including the primers shown in SEQ ID NO: 21 and 22; and a second probe for detecting mutations in the hot regions of KRAS G12 and G13 used in combination with the second primer pair, the second probe being a combination of the following probes: the probe shown in SEQ ID NO: 31, the probe shown in SEQ ID NO: 34, the probe shown in SEQ ID NO: 36, the probe shown in SEQ ID NO:

39.

2. The reagent according to claim 1, wherein The reagent further includes: (c) A third primer pair for detecting TERT promoter C228T mutation and C250T mutation, the third primer pair including the primers shown in SEQ ID NO: 9 and 10; and a third probe for detecting TERT promoter C228T mutation and C250T mutation used in combination with the third primer pair, the third probe being a combination of the following probes: the probe shown in SEQ ID NO: 19, the probe shown in SEQ ID NO:

20.

3. The reagent according to claim 1, wherein The mutations in the hot regions of KRAS G12 and G13 include the following mutations: (1) Glycine G at the 12th position of the amino acid sequence of KRAS protein is mutated to alanine A (i.e., G12A); (2) Glycine G at the 12th position of the amino acid sequence of KRAS protein is mutated to cysteine C (i.e., G12C); (3) Glycine G at the 12th position of the amino acid sequence of KRAS protein is mutated to aspartic acid D (i.e., G12D); (4) Glycine G at the 12th position of the amino acid sequence of KRAS protein is mutated to serine S (i.e., G12S); (5) Glycine G at the 12th position of the amino acid sequence of KRAS protein is mutated to arginine R (i.e., G12R); (6) Glycine G at the 12th position of the amino acid sequence of KRAS protein is mutated to valine V (i.e., G12V); (7) Glycine G at the 13th position of the amino acid sequence of KRAS protein is mutated to cysteine C (i.e., G13C); (8) Glycine G at the 13th position of the amino acid sequence of KRAS protein is mutated to aspartic acid D (i.e., G13D).

4. The reagent according to claim 1, wherein The sequence of SEQ ID NO: 7 is: CGAGATTTC+T+CTGTAGCT; the sequence of SEQ ID NO: 8 is: TCGAGATTT+C+ACTGTAGC; the sequence of SEQ ID NO: 31 is: AGCT+G+GT+G+GCG; the sequence of SEQ ID NO: 34 is: AGCT+G+a+TGGCGTAgg; the sequence of SEQ ID NO: 36 is: tggAGC+T+A+GTGGCGT; the sequence of SEQ ID NO: 39 is: AGCTGG+TG+AC+GTAgg; In each formula, "+T" represents locked nucleic acid T, "+C" represents locked nucleic acid C, and "+A" represents locked nucleic acid A.

5. The reagent according to claim 1, wherein The structure of the first probe from 5'-3' is shown in formula (I): Z1-Z2-Z3 (I) Wherein, Z1 is a fluorophore; Z2 is a specific complementary nucleic acid sequence containing or not containing locked nucleic acids; Z3 is a quencher; "-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.

6. The reagent according to claim 1, wherein The structure of the second probe from 5'-3' is shown in formula (I): Z1'-Z2'-Z3' (II) Wherein, Z1' is a fluorophore; Z2' is a specific complementary nucleic acid sequence containing or not containing locked nucleic acids; Z3' is a quencher; "-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.

7. A kit, characterized in that, The kit contains the reagent for detecting gene mutations as claimed in claim 1.

8. The kit according to claim 7, wherein The primers and probes in the reagent exist in the form of a primer-probe mixture.

9. The kit according to claim 7, wherein, The kit further includes a positive control product and a negative control product.

10. A method for non-diagnostically detecting whether a sample to be tested contains a gene mutation in vitro, characterized in that, It includes the steps: (s1) Provide a PCR reaction system, which contains a sample to be tested as a template and the reagent for detecting gene mutations as claimed in claim 1; (s2) Perform a PCR reaction on the PCR reaction system in step (S1) to obtain an amplification product; (s3) Analyze the amplification product generated in step (S2) to obtain the analysis result of whether the sample to be tested contains gene mutations.

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