A digital PCR detection reagent for BRAF, KRAS and TERT gene mutations and its application

Abstract

The present invention provides a digital PCR detection reagent and application for BRAF, KRAS, and TERT gene mutations. Specifically, preferred primer pairs, amplification methods, nucleic acid probes, and detection systems targeting sequences containing BRAF, KRAS, and TERT gene mutations are provided. Furthermore, a kit for detecting BRAF, KRAS, and TERT gene mutations is provided. Through optimized primer pairs and probes, as well as corresponding reaction conditions, the present invention can detect BRAF, KRAS, and TERT gene mutations with high sensitivity and specificity in different samples.

Description

Technical Field

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

[0002] In recent years, the incidence of thyroid cancer has shown a significant upward trend. Although differentiated thyroid cancer (DTC) has a generally favorable prognosis, some cases still face clinical challenges such as aggressive growth, metastasis, and iodine-refractory disease. Studies have shown that the synergistic effects of BRAF V600E, KRAS, and TERT promoter gene mutations are closely associated with the development, pathological classification, and treatment resistance of thyroid cancer. As a core molecular marker for papillary thyroid cancer (PTC), the presence of BRAF V600E mutation is significantly associated with increased risk of tumor invasion, lymph node metastasis, and postoperative recurrence. KRAS mutations exhibit specific activation characteristics in follicular thyroid cancer (FTC) and poorly differentiated cancers, suggesting that they may be involved in the abnormal regulation of the MAPK / ERK signaling pathway. TERT promoter mutations have been shown to be an independent predictor of thyroid cancer dedifferentiation and poor prognosis, and are strongly correlated with shortened overall survival.

[0003] Current clinical testing systems mostly utilize single-gene, step-by-step testing strategies, which present technical bottlenecks such as long testing cycles (>72 hours), high sample consumption (requiring ≥3 serial sections), and insufficient sensitivity (conventional PCR methods have a detection limit of only 1-5%). Especially for thyroid nodules with uncertain diagnosis based on fine needle aspiration cytology (FNA), existing technologies struggle to simultaneously obtain multi-dimensional molecular typing information from tiny sample volumes, resulting in the inability to achieve accurate risk stratification in approximately 30% of cases. Furthermore, while next-generation sequencing (NGS) platforms can enable multi-gene testing, their operational complexity, high costs, and data analysis delays severely limit widespread clinical application.

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

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

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

[0007] In a first aspect of the present invention, a reagent for detecting a gene mutation is provided, the reagent comprising:

[0008] (a) a first primer pair for detecting a BRAF V600E mutation, the first primer pair comprising primers set forth in SEQ ID NOs: 1 and 2; and a first probe for detecting a BRAF V600E mutation used in conjunction with the first primer pair, the first probe being a combination of the following probes: a probe set forth in SEQ ID NO: 7 and a probe set forth in SEQ ID NO: 8; and

[0009] (b) a second primer pair for detecting mutations in the KRAS G12 and G13 hotspot regions, the second primer pair comprising primers set forth in SEQ ID NOs: 21 and 22; and a second probe used in conjunction with the second primer pair for detecting mutations in the KRAS G12 and G13 hotspot regions, the second probe being a combination of the following probes: a probe set forth in SEQ ID NO: 31, a probe set forth in SEQ ID NO: 34, a probe set forth in SEQ ID NO: 36, and a probe set forth in SEQ ID NO: 39.

[0010] In another preferred embodiment, the reagent further comprises:

[0011] (c) a third primer pair for detecting TERT promoter C228T mutation and C250T mutation, the third primer pair comprising primers shown in SEQ ID NOs: 9 and 10; and a third probe for detecting TERT promoter C228T mutation and C250T mutation used in conjunction with the third primer pair, the third probe being a combination of the following probes: the probe shown in SEQ ID NO: 19 and the probe shown in SEQ ID NO: 20.

[0012] In another preferred embodiment, 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 (ie, V600E).

[0013] In another preferred embodiment, the KRAS G12 and G13 hotspot mutations include the following mutations:

[0014] (1) The glycine G at position 12 of the KRAS protein amino acid sequence mutates to an alanine A (i.e., G12A);

[0015] (2) The glycine G at position 12 of the KRAS protein amino acid sequence mutates to a cysteine ​​C (i.e., G12C);

[0016] (3) The glycine G at position 12 of the KRAS protein amino acid sequence mutates to aspartic acid D (i.e., G12D);

[0017] (4) The glycine G at position 12 of the KRAS protein amino acid sequence mutates to serine S (i.e., G12S);

[0018] (5) The glycine G at position 12 of the KRAS protein amino acid sequence mutates to arginine R (i.e., G12R);

[0019] (6) The glycine G at position 12 of the KRAS protein amino acid sequence mutates to valine V (i.e., G12V);

[0020] (7) The glycine G at position 13 of the KRAS protein amino acid sequence mutates to cysteine ​​C (i.e., G13C);

[0021] (8) The glycine G at position 13 of the KRAS protein amino acid sequence mutates to aspartic acid D (i.e., G13D).

[0022] In another preferred embodiment, the TERT promoter C228T mutation refers to the mutation of cytosine C at position 228 of the TERT promoter gene nucleotide sequence to thymine T (TERT c.228 C>T); the TERT promoter C250T mutation refers to the mutation of cytosine C at position 250 of the TERT promoter gene nucleotide sequence to thymine T (TERT c.250 C>T.

[0023] In another preferred embodiment, the sequence of SEQ ID NO: 7 is: CGAGATTTC+T+CTGTAGCT; the sequence of SEQ ID NO: 8 is: TCGAGATTT+C+ACTGTAGC;

[0024] In each formula, “+T” represents locked nucleotide T, “+C” represents locked nucleotide C, and “+A” represents locked nucleotide A.

[0025] In another preferred embodiment, 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;

[0026] In each formula, “+T” represents locked nucleotide T, “+G” represents locked nucleotide G, and “+A” represents locked nucleotide A.

[0027] In another preferred embodiment, the sequence of SEQ ID NO: 19 is: ACCCC+T+C+CCGGG; the sequence of SEQ ID NO: 20 is: AGCCC+C+C+TCCGG;

[0028] In each formula, “+T” represents locked nucleotide T, and “+C” represents locked nucleotide C.

[0029] In another preferred embodiment, the 5'-3' structure of the first probe is as shown in formula (I):

[0030] Z1-Z2-Z3 (I)

[0031] in,

[0032] Z1 is a fluorescent group;

[0033] Z2 is a specific complementary nucleic acid sequence containing or not containing locked nucleotides;

[0034] Z3 is a quenching group;

[0035] “-” represents a chemical bond, a linker, or a linker consisting of 1-3 nucleotides.

[0036] In another preferred embodiment, the Z2-specific nucleic acid sequence targets the wild-type BRAF V600 site.

[0037] In another preferred embodiment, the Z2-specific nucleic acid sequence targets the mutant BRAF V600E site.

[0038] In another preferred embodiment, the Z2 contains a locked nucleotide modification.

[0039] In another preferred embodiment, the modification site of the locked nucleic acid modification covers the mutated base and 2-4 nucleotides upstream and downstream thereof.

[0040] In another preferred embodiment, the sequence of Z2 is selected from the following group:

[0041] Sequence shown in SEQ ID NO: 7: CGAGATTTC+T+CTGTAGCT;

[0042] Sequence shown in SEQ ID NO: 8: TCGAGATTT+C+ACTGTAGC;

[0043] In each formula, “+T” represents locked nucleotide T, “+C” represents locked nucleotide C, and “+A” represents locked nucleotide A.

[0044] In another preferred embodiment, the fluorescent groups are independently located at the 5' end, 3' end and the middle of the nucleic acid probe.

[0045] In another preferred embodiment, the fluorescent group and the quencher group are independently located at the 5' end, the 3' end, and / or the middle.

[0046] In another preferred embodiment, the fluorescent group includes a fluorescent group cross-linked with a DNA probe.

[0047] In another preferred embodiment, the fluorescent group is selected from the following group: FAM, HEX, ROX, VIC, FITC, BODIPY-FL, G-Dye100, FluorX, Cy3, Cy5, Cy5.5, Texas Red, or a combination thereof.

[0048] 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.

[0049] In another preferred embodiment, the 5'-3' structure of the second probe is as shown in formula (I):

[0050] Z1'-Z2'-Z3' (II)

[0051] in,

[0052] Z1′ is a fluorescent group;

[0053] Z2' is a specific complementary nucleic acid sequence containing or not containing locked nucleotides;

[0054] Z3' is a quenching group;

[0055] “-” represents a chemical bond, a linker, or a linker consisting of 1-3 nucleotides.

[0056] In another preferred embodiment, the 3' end of the probe shown in SEQ ID NO: 31 is blocked.

[0057] In another preferred embodiment, the 3' end blocking treatment method includes: 3' end phosphorylation and C3 spacer.

[0058] 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.

[0059] In another preferred embodiment, the Z2'-specific nucleic acid sequence targets a site selected from the group consisting of:

[0060] (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.

[0061] In another preferred embodiment, the Z2' contains a locked nucleotide modification.

[0062] In another preferred embodiment, the modification site of the locked nucleic acid modification covers the mutated base and 2-4 nucleotides upstream and downstream thereof.

[0063] In another preferred embodiment, the sequence of Z2 is selected from the following group:

[0064] Sequence shown in SEQ ID NO: 31: AGCT+G+GT+G+GCG;

[0065] Sequence shown in SEQ ID NO: 34: AGCT+G+a+TGGCGTAgg;

[0066] Sequence shown in SEQ ID NO: 36: tggAGC+T+A+GTGGCGT;

[0067] Sequence shown in SEQ ID NO: 39: AGCTGG+TG+AC+GTAgg;

[0068] In each formula, “+T” represents locked nucleotide T, “+G” represents locked nucleotide G, and “+A” represents locked nucleotide A.

[0069] In another preferred embodiment, the structure 5'-3' of the third probe is as shown in formula (I):

[0070] Z1''-Z2''-Z3'' (III)

[0071] in,

[0072] Z1′′ is a fluorescent group;

[0073] Z2'' is a specific complementary nucleic acid sequence containing or not containing locked nucleotides;

[0074] Z3'' is a quenching group;

[0075] “-” represents a chemical bond, a linker, or a linker consisting of 1-3 nucleotides.

[0076] 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.

[0077] In another preferred embodiment, the Z2″-specific nucleic acid sequence targets the mutant TERT C228T site and / or the wild-type TERT C250T site.

[0078] In another preferred embodiment, the Z2″ contains a locked nucleotide modification.

[0079] In another preferred embodiment, the modification site of the locked nucleic acid modification covers the mutated base and 2-4 nucleotides upstream and downstream thereof.

[0080] In another preferred embodiment, the sequence of Z2'' is selected from the following group:

[0081] Sequence shown in SEQ ID NO: 19: ACCCC+T+C+CCGGG;

[0082] Sequence shown in SEQ ID NO: 20: AGCCC+C+C+TCCGG;

[0083] In each formula, “+T” represents locked nucleotide T, and “+C” represents locked nucleotide C.

[0084] In a second aspect of the present invention, a kit is provided, wherein the kit contains the reagent for detecting gene mutation according to the first aspect of the present invention.

[0085] In another preferred embodiment, the primers and probes in the reagents exist in the form of a primer-probe mixture.

[0086] In another preferred embodiment, the kit further comprises a positive quality control product and a negative quality control product.

[0087] In another preferred example, the positive quality control materials are: a mixture of genomic DNA of the BRAF V600E mutant cell line RKO and genomic DNA of the wild-type cell line 293T (mutation rate 3%), a mixture of genomic DNA of the TERT promoter C228T mutant cell line HepG and genomic DNA of the wild-type cell line 293T (mutation rate 1%), a mixture of artificially synthesized TERT promoter C250T mutant plasmid and genomic DNA of the wild-type cell line 293T (mutation rate 1%), and a mixture of genomic DNA of the KRAS G12 / G13 hotspot mutant cell lines Sw1116, A549, CAL-62, HCT-116, NCL-H1734 and artificially synthesized plasmids with genomic DNA of the wild-type cell line 293T (mutation rate 1-3%); the concentration of each is about 3000 copies / ul.

[0088] In another preferred embodiment, the negative control material is: wild-type cell line 293T genomic DNA (concentration of about 3000 copies / ul).

[0089] In a third aspect of the present invention, a method for non-diagnostic in vitro detection of whether a sample contains a gene mutation is provided, comprising the steps of:

[0090] (s1) providing a PCR reaction system, wherein the PCR reaction system contains a sample to be tested as a template and the reagent for detecting gene mutations according to the first aspect of the present invention;

[0091] (s2) performing a PCR reaction on the PCR reaction system of step (S1) to obtain an amplified product;

[0092] (s3) Analyzing the amplified product produced in step (S2) to obtain an analysis result of whether the sample to be tested contains a gene mutation.

[0093] In another preferred embodiment, the reagent further comprises:

[0094] (c) a third primer pair for detecting TERT promoter C228T mutation and C250T mutation, the third primer pair comprising primers shown in SEQ ID NOs: 9 and 10; and a third probe for detecting TERT promoter C228T mutation and C250T mutation used in conjunction with the third primer pair, the third probe being a combination of the following probes: the probe shown in SEQ ID NO: 19 and the probe shown in SEQ ID NO: 20.

[0095] In another preferred embodiment, the sample to be tested is selected from the following group: formalin-fixed paraffin-embedded (FFPE) tissue section sample, fine needle aspiration (FNA) cytology sample, or a combination thereof.

[0096] In another preferred embodiment, the sample requirement is that the thickness of a single slice is ≤5 μm or the number of cells is ≥100.

[0097] In another preferred embodiment, in step (s1), the reaction system further contains: 5-20% glycerol and 0.01-1% Poloxamer 188.

[0098] In another preferred embodiment, in step (s1), the reaction system further contains: 10% glycerol and 0.5% Poloxamer 188.

[0099] In another preferred embodiment, in the reaction system, the probes for detecting BRAF V600E mutation include a wild-type probe for detecting the BRAF V600 site and a mutant probe for detecting the BRAF V600E site; and the fluorescent reporter group of the wild-type probe is HEX, and the fluorescent reporter group of the mutant probe is FAM.

[0100] In another preferred embodiment, in the reaction system, the probes for detecting mutations in the KRAS G12 and G13 hotspot regions include wild-type probes for detecting KRAS G12 and G13 sites and mutant probes for detecting mutation sites in the KRAS G12 and G13 hotspot regions; the wild-type probes have no fluorescent reporter group at the 5' end and are phosphorylated and blocked at the 3' end; the fluorescent reporter groups of the mutant probes are all Cy5.5.

[0101] In another preferred example, the probes for detecting the C228T mutation and the C250T mutation of the TERT promoter include a first wild-type probe for detecting the C228 site of the TERT promoter and a second wild-type probe for detecting the C250 site of the TERT promoter; the fluorescent reporter group of the first wild-type probe is Cy5, and the fluorescent reporter group of the second wild-type probe is ROX.

[0102] In another preferred embodiment, the analysis result is a qualitative result.

[0103] In another preferred embodiment, the PCR reaction system is a digital PCR reaction system.

[0104] In another preferred embodiment, in the reagent, the probes for detecting BRAF and KRAS mutant genes use different fluorescent reporter groups.

[0105] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0106] In another preferred embodiment, the method is an in vitro method.

[0107] In another preferred embodiment, the detection sensitivity of the method is 0.1% mutant allele frequency (MAF).

[0108] In another preferred embodiment, the detection limit of the method is 5 copies / reaction system of mutant DNA.

[0109] In another preferred embodiment, the detection period of the method is ≤ 2 hours.

[0110] In a fourth aspect of the present invention, there is provided a use of the reagent for detecting mutations according to the first aspect of the present invention or the kit according to the second aspect of the present invention for preparing a diagnostic product, wherein the diagnostic product is used for:

[0111] (a) Predicting tumor aggressiveness and postoperative recurrence risk (high-risk criteria: presence of ≥2 gene mutations);

[0112] (b) Screening of cases sensitive to targeted therapy with lenvatinib and sorafenib (mutation abundance threshold ≥ 5%).

[0113] In another preferred embodiment, when ≥2 gene mutations are present simultaneously, the subject is judged to have a high risk of tumor invasion or postoperative recurrence.

[0114] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1 The amplification effects of the three pairs of primers used to specifically amplify the BRAF V600E mutation site in Example 1 are shown (PCR electrophoresis results of primer screening).

[0116] Figure 2 The figure shows a two-dimensional digital PCR image of the primers P1-F / R combined with the probe to detect BRAF V600E in Example 1.

[0117] Figure 3 The figure shows a two-dimensional digital PCR image of the primers P2-F / R combined with the probe to detect BRAF V600E in Example 1.

[0118] Figure 4 The figure shows a two-dimensional digital PCR image of the primers P3-F / R combined with the probe to detect BRAF V600E in Example 1.

[0119] Figure 5 The amplification effects of the five primer pairs used to specifically amplify the TERT promoter mutation position in Example 2 are shown.

[0120] Figure 6 A two-dimensional digital PCR image of the wild-type TERT promoter detected using the selected primer pairs and probes in Example 2 is shown.

[0121] Figure 7 The figure shows a two-dimensional digital PCR diagram for detecting the wild-type TERT promoter after optimizing the reaction system in Example 3.

[0122] Figure 8 The figure shows a two-dimensional digital PCR diagram for detecting the TERT promoter C228T mutation after optimizing the reaction system in Example 3.

[0123] Figure 9 The figure shows a two-dimensional digital PCR diagram for detecting the TERT promoter C250T mutation after optimizing the reaction system in Example 3.

[0124] Figure 10 The amplification effects of the five primer pairs used in Example 4 for specific amplification of the KRAS G12 / G13 hotspot region are shown.

[0125] Figure 11 A two-dimensional graph of digital PCR for detecting KRAS G12A mutation in Example 5 is shown.

[0126] Figure 12 A two-dimensional graph of digital PCR for detecting KRAS G12C mutation in Example 5 is shown.

[0127] Figure 13 A two-dimensional graph of digital PCR for detecting KRAS G12D mutation in Example 5 is shown.

[0128] Figure 14 A two-dimensional graph of digital PCR for detecting KRAS G12S mutation in Example 5 is shown.

[0129] Figure 15 A two-dimensional graph of digital PCR for detecting KRAS G12R mutation in Example 5 is shown.

[0130] Figure 16 A two-dimensional graph of digital PCR for detecting KRAS G12V mutation in Example 5 is shown.

[0131] Figure 17 A two-dimensional graph of digital PCR for detecting KRAS G13C mutation in Example 5 is shown.

[0132] Figure 18 A two-dimensional graph of digital PCR for detecting KRAS G13D mutation in Example 5 is shown.

[0133] Figure 19A two-dimensional graph of digital PCR for detecting KRAS G12A mutation in Example 6 is shown.

[0134] Figure 20 A two-dimensional graph of digital PCR for detecting KRAS G12C mutation in Example 6 is shown.

[0135] Figure 21 A two-dimensional graph of digital PCR for detecting KRAS G12D mutation in Example 6 is shown.

[0136] Figure 22 A two-dimensional graph of digital PCR for detecting KRAS G12S mutation in Example 6 is shown.

[0137] Figure 23 A two-dimensional graph of digital PCR for detecting KRAS G12R mutation in Example 6 is shown.

[0138] Figure 24 A two-dimensional graph of digital PCR for detecting KRAS G12V mutation in Example 6 is shown.

[0139] Figure 25 A two-dimensional graph of digital PCR for detecting KRAS G13C mutation in Example 6 is shown.

[0140] Figure 26 A two-dimensional graph of digital PCR for detecting KRAS G13D mutation in Example 6 is shown. DETAILED DESCRIPTION

[0141] After extensive and in-depth research, the inventors have discovered that through extensive screening, particularly by optimizing primer and probe sequences, combined with a digital PCR platform, they have effectively improved the effectiveness of gene mutation detection. This method overcomes the technical bottlenecks of existing gene mutation detection technologies, such as long detection cycles, high sample consumption, and insufficient detection sensitivity. They have provided a method with high specificity, high sensitivity, and strong anti-interference capabilities for the simultaneous detection of BRAF, KRAS, and TERT gene mutations. Based on this, the inventors have completed the present invention.

[0142] 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 mutation genes. By designing and optimizing primer and probe sequences, a primer and probe combination that can simultaneously detect the above three gene mutations without interfering with each other in the same reaction is finally screened and obtained, and a digital PCR detection system is established to qualitatively and quantitatively detect the mutations of the above genes. The method and reagent of the present invention have unexpectedly high sensitivity and high specificity when used to detect the above gene mutations, and can detect samples of different difficulty levels.

[0143] the term

[0144] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0145] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition 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.).

[0146] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0147] Sequence identity is determined by comparing the 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 at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

[0148] Digital PCR technology

[0149] Digital PCR technology is based on the single-molecule PCR method to perform nucleic acid quantification by counting, and is an absolute quantitative method. It mainly uses microfluidics or droplet methods, which are currently popular research fields in analytical chemistry, to disperse a large amount of diluted nucleic acid solution into microreactors or droplets on the chip, with the number of nucleic acid templates in each reactor being less than or equal to 1. In this way, after the PCR cycle, the fluorescence signal of each droplet is analyzed after the amplification is completed. The reactor with nucleic acid molecule template will give a fluorescent signal, and the reactor without template will not give a fluorescent signal. Based on the relative proportion and the volume of the reactor, the nucleic acid concentration of the original solution can be calculated.

[0150] Compared with conventional qPCR, digital PCR can accurately quantitatively analyze 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 perform different tests at the same time. 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.

[0151] In digital PCR, droplets containing a sample gene template prepared for dilution to an average copy number of 0.5-1, amplification primers, and a fluorescent probe are dispensed into individual wells, and microemulsion PCR is performed. Wells that display a fluorescent signal are then counted as "1" because a sample with a gene copy number of 1 was dispensed into that well and displayed a fluorescent signal after amplification. Wells that do not display a signal are counted as "0" because a sample with a gene copy number of 0 was dispensed into that well and, due to lack of amplification, did not display a fluorescent signal. This approach enables absolute quantification.

[0152] Primers

[0153] A primer is a macromolecule with a specific nucleotide sequence that stimulates the synthesis of nucleotides at the initiation of nucleotide polymerization and is covalently linked to the reactants. Primers are typically two synthetic 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 a second DNA template strand at the other end of the target region.

[0154] In the present invention, in order to improve the sensitivity of the detection system, the corresponding gene fragment in the detection system is pre-amplified, and primers corresponding to the sequence where the mutation is located are designed.

[0155] In a preferred example, for the sequence where BRAF V600E is located, multiple pairs of primers were designed upstream and downstream of the BRAF gene V600 site. After experimental testing, the primer pair was finally determined to be SEQ ID NO: 1 and 2; for the sequence where TERT promoter C228T and C250T are located, multiple pairs of primers were designed upstream and downstream of the TERT gene promoter C228 and C250 sites, respectively. After testing, the optimal primer pair was 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 were designed upstream and downstream of the KRAS gene G12 and G13 sites, respectively. After experimental testing, the optimal primer pair was finally determined to be SEQ ID NO: 21 and 22.

[0156] probe

[0157] In this article, "probe", "nucleic acid probe" and "gene probe" are interchangeable 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 to generate a hybridization signal, which can display the target gene from the vast genome. According to the hybridization principle, the nucleic acid sequence used as a probe must meet at least 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 easy to detect. The nucleic acid probe can include the entire gene or only a part of the gene; it can be DNA itself or RNA transcribed from it. In the present invention, the probe also refers to a modified primer, which has chemical modification groups at both ends or in the middle of the modified primer. These chemical modifications have special functions including but not limited to: signal indication, enhanced connection with reactants, etc.

[0158] In another preferred embodiment, the 5'-3' structure of the first probe is as shown in formula (I):

[0159] Z1-Z2-Z3 (I)

[0160] in,

[0161] Z1 is a fluorescent group;

[0162] Z2 is a specific complementary nucleic acid sequence containing or not containing locked nucleotides;

[0163] Z3 is a quenching group;

[0164] “-” represents a chemical bond, a linker, or a linker consisting of 1-3 nucleotides.

[0165] In another preferred embodiment, the Z2-specific nucleic acid sequence targets the wild-type BRAF V600 site.

[0166] In another preferred embodiment, the Z2-specific nucleic acid sequence targets the mutant BRAF V600E site.

[0167] In another preferred embodiment, the Z2 contains a locked nucleotide modification.

[0168] In another preferred embodiment, the sequence of Z2 is selected from the following group:

[0169] Sequence shown in SEQ ID NO: 7: CGAGATTTC+T+CTGTAGCT;

[0170] Sequence shown in SEQ ID NO: 8: TCGAGATTT+C+ACTGTAGC;

[0171] In each formula, “+T” represents locked nucleotide T, “+C” represents locked nucleotide C, and “+A” represents locked nucleotide A.

[0172] In another preferred embodiment, the fluorescent groups are independently located at the 5' end, 3' end and the middle of the nucleic acid probe.

[0173] In another preferred embodiment, the fluorescent group and the quencher group are independently located at the 5' end, the 3' end, and / or the middle.

[0174] In another preferred embodiment, the fluorescent group includes a fluorescent group cross-linked with a DNA probe.

[0175] In another preferred embodiment, the fluorescent group is selected from the following group: FAM, HEX, ROX, VIC, FITC, BODIPY-FL, G-Dye100, FluorX, Cy3, Cy5, Cy5.5, Texas Red, or a combination thereof.

[0176] 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.

[0177] In another preferred embodiment, the 5'-3' structure of the second probe is as shown in formula (I):

[0178] Z1'-Z2'-Z3' (II)

[0179] in,

[0180] Z1′ is a fluorescent group;

[0181] Z2' is a specific complementary nucleic acid sequence containing or not containing locked nucleotides;

[0182] Z3' is a quenching group;

[0183] “-” represents a chemical bond, a linker, or a linker consisting of 1-3 nucleotides.

[0184] 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.

[0185] In another preferred embodiment, the Z2'-specific nucleic acid sequence targets a site selected from the group consisting of:

[0186] (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.

[0187] In another preferred embodiment, the Z2' contains a locked nucleotide modification.

[0188] In another preferred embodiment, the sequence of Z2 is selected from the following group:

[0189] Sequence shown in SEQ ID NO: 31: AGCT+G+GT+G+GCG;

[0190] Sequence shown in SEQ ID NO: 34: AGCT+G+a+TGGCGTAgg;

[0191] Sequence shown in SEQ ID NO: 36: tggAGC+T+A+GTGGCGT;

[0192] Sequence shown in SEQ ID NO: 39: AGCTGG+TG+AC+GTAgg;

[0193] In each formula, “+T” represents locked nucleotide T, “+G” represents locked nucleotide G, and “+A” represents locked nucleotide A.

[0194] In another preferred embodiment, the structure 5'-3' of the third probe is as shown in formula (I):

[0195] Z1''-Z2''-Z3'' (III)

[0196] in,

[0197] Z1′′ is a fluorescent group;

[0198] Z2'' is a specific complementary nucleic acid sequence containing or not containing locked nucleotides;

[0199] Z3'' is a quenching group;

[0200] “-” represents a chemical bond, a linker, or a linker consisting of 1-3 nucleotides.

[0201] 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.

[0202] In another preferred embodiment, the Z2″-specific nucleic acid sequence targets the mutant TERT C228T site and / or the wild-type TERT C250T site.

[0203] In another preferred embodiment, the Z2″ contains a locked nucleotide modification.

[0204] In another preferred embodiment, the sequence of Z2'' is selected from the following group:

[0205] Sequence shown in SEQ ID NO: 19: ACCCC+T+C+CCGGG;

[0206] Sequence shown in SEQ ID NO: 20: AGCCC+C+C+TCCGG;

[0207] In each formula, “+T” represents locked nucleotide T, and “+C” represents locked nucleotide C.

[0208] Modification of primers and probes

[0209] In the present invention, the nucleic acid sequence of the primer includes an unmodified or modified primer sequence.

[0210] Preferably, the present inventors modified the probe with locked nucleotides (LNA), which can significantly improve the specificity of the probe, thereby improving the sensitivity and specificity of the detection results.

[0211] In a preferred embodiment of the present invention, the modification method is selected from: phosphorylation, biotin, digoxigenin, internal amino modification, 5' amino modification, 3' amino modification, thiol, spacer, thiolate, deoxyuridine (dU), deoxyinosine (dI), or a combination thereof.

[0212] Phosphorylation: 5' phosphorylation is used in linkers, cloning, and gene construction, as well as in ligase-catalyzed ligation reactions. 3' phosphorylation provides resistance to 3' exonuclease digestion and is also used to block DNA polymerase-catalyzed DNA chain extension reactions.

[0213] Biotin modification: Primers are biotin-labeled and can be used for non-radioactive immunoassays to detect proteins, intracellular chemical staining, cell separation, nucleic acid separation, hybridization detection of specific DNA / RNA sequences, ion channel conformational changes, etc.

[0214] Digoxin modification: Digoxin is linked to the C5 position of uracil via an 11-atom spacer. Hybridized Digoxin probes can be detected by anti-Digoxin antibodies. Digoxin-labeled probes can be used in various hybridization reactions, such as DNA-DNA hybridization (Southern blotting), DNA-RNA hybridization (Northern blotting), dot blotting, clonal hybridization, in situ hybridization, and enzyme-linked immunosorbent assay (ELISA).

[0215] Internal amino modification: C6-dT aminolinker is primarily used to add internal modification to thymine residues. The modified amino group is 10 atoms away from the backbone, allowing for further labeling and enzyme ligation (e.g., alkaline phosphatase). Currently, internal amino modification-mediated dT-Dabcyl, dT-Biotin, and dT-Digoxingenin modifications are available.

[0216] 5' amino modification: This can be used to prepare functionalized oligonucleotides and is widely used in DNA microarrays and multiplexed diagnostic systems. Currently, two types of modifications are available: the 5' C6 amino modification and the 5' C12 amino modification. The 5' C6 amino modification can be used to attach compounds that can be placed in close proximity to the oligonucleotide without affecting its function. The 5' C12 amino modification is used for attaching affinity purification groups and for some fluorescent labels, particularly when fluorescence may be quenched by proximity to the DNA strand.

[0217] 3' Amino Modification: Currently available is a 3' C6 amino modification. This 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 modified with an amino group for alternative ligations. Furthermore, the 3' modification can inhibit 3' exonuclease digestion, making it useful in antisense experiments.

[0218] Thiol modification: 5'-thiol modification is similar in many respects to amino modification. Thiol groups can be used to attach various modifications, such as fluorescent markers and biotin. For example, thiol-linked fluorescent probes can be prepared in the presence of iodoacetic acid and maleimide derivatives. 5'-thiol modification is primarily achieved using 5'-thiol-modifier monomers (5'-Thiol-Modifier C6-CE Phosphoramidite or Thiol-Modifier C6 SS CE Phosphoramidite). Modification with the 5'-Thiol-Modifier C6-CE monomer requires silver nitrate oxidation to remove the trityl protecting group, while modification with the Thiol-Modifier C6 SS CE monomer requires DTT to reduce the disulfide bond to the thiol group.

[0219] Spacer modification: Spacers provide the necessary spacing for oligonucleotide labeling, reducing interactions between the labeling group and the oligonucleotide. They are primarily used to study DNA hairpin structures and duplex structures. The C3 spacer is primarily used to mimic the three-carbon spacer between the 3' and 5' hydroxyl groups of the ribose sugar or to "replace" an unknown base in a sequence. The 3'-Spacer C3 is used to introduce a 3' spacer to prevent the action of 3' exonucleases and 3' polymerases. Spacer 18 is often used to introduce a strongly hydrophilic group.

[0220] Thio-modified oligonucleotides are primarily used in antisense experiments to protect against nuclease degradation. While fully thio-modified oligonucleotides are acceptable, increasing the number of thio-modified bases decreases the Tm of the oligonucleotide. To mitigate this effect, thio-modify 2-5 bases at each end of the primer, typically selecting 3 bases at both the 5' and 3' ends.

[0221] Deoxyuracil modification: Deoxyuracil can be inserted into oligonucleotides to increase the melting point of the duplex, thereby increasing duplex stability. Each deoxythymine replaced by deoxyuracil increases the duplex melting point by 1.7°C.

[0222] Deoxyhypoxanthine modification: Deoxyhypoxanthine is a naturally occurring base. While not a true universal base, it is relatively more stable when combined with other bases than with other base mismatches. The binding affinity of deoxyhypoxanthine with other bases is dI:dC > dI:dA > dI:dG > dI:dT. Under the catalysis of DNA polymerase, deoxyhypoxanthine preferentially binds to dC.

[0223] The main advantages of the present invention include:

[0224] (a) The present invention uses Taqman probes in combination with digital PCR to address problems such as low sensitivity, poor specificity, high requirements for sample type and quality, and complex positive interpretation methods.

[0225] (b) High Sensitivity: Because this method utilizes a digital PCR platform, it can divide the reaction system into approximately 20,000 micro-reactions, theoretically capable of detecting single-copy mutations, offering a sensitivity advantage unmatched by other technologies. The detection method of this invention has been validated to achieve a minimum detection limit of 5 copies / reaction.

[0226] (c) Strong specificity: The designed specific primers and probes are targeted at the specific sequences of the mutation and can specifically amplify the target location.

[0227] (d) The detection reagent or detection method of the present invention has loose requirements on sample type and quality and is highly resistant to interference. Due to the high sensitivity of the present invention, applicable sample types include FNA (which is relatively easy to obtain but the sample quantity is small).

[0228] (e) Simple positive interpretation method: Since the present invention uses an absolute quantitative method, there is no need to set up a control standard curve or compare with an internal reference. The results can be determined based on the two-dimensional fluorescence graph to determine whether the target mutant template is present (Table A).

[0229] Table A Test results table

[0230]

[0231] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed 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 indicated, percentages and parts are by weight.

[0232] Example 1 Design and screening of BRAF V600E mutation-specific primers and probes

[0233] 1.1 Materials and Methods

[0234] (1) Sample source:

[0235] Positive control: genomic DNA from the BRAF V600E mutant cell line RKO;

[0236] Negative control: genomic DNA from the BRAF wild-type cell line 293T.

[0237] (2) Primer / probe design:

[0238] Based on the BRAF gene c.1799T>A mutation site and primer-probe design principles, three pairs of candidate primers (primer P1-primer P3) and two modified probes were designed, as shown in Table 1. The probes were modified with locked nucleotides (LNA) to cover the mutation site and several bases upstream and downstream. The quencher 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.

[0239] Table 1

[0240]

[0241] Note: “+A” indicates locked nucleotide A, “+T” indicates locked nucleotide T, and “+C” indicates locked nucleotide C.

[0242] (3) Preliminary screening:

[0243] Primer screening: Prepare PCR reaction system: 5×HS Taq Buffer with Mg 2+ 4μL, dNTPs (10mM each) 0.5μL, HotStart Taq DNA Polymerase 0.2μL, F primer (10μM) 1μL, R primer (100μM) 1μL, template 2μL, add water to make up to 20μL.

[0244] Cycling conditions: 95°C for 10 min, 40 cycles (95°C for 30 s, 56°C for 15 s, 72°C for 15 s).

[0245] Detection was performed by electrophoresis of 10 μL PCR product.

[0246] The amplification effect of three pairs of primers ( Figure 1 ) are all acceptable and subsequent digital PCR experiments are performed.

[0247] (4) Digital PCR experiment:

[0248] Prepare PCR reaction system: 5× HS Taq Buffer with Mg 2+ 6μL, dNTPs (10mM each) 0.75μL, HotStart Taq DNA Polymerase 0.2μL, F primer (10μM) 1μL, R primer (10μM) 1μL, WT probe (10μM) 0.5μL, MT probe (10μM) 0.5μL, template (wild type or mutant) 2μL, add water to make up to 30μL.

[0249] Follow the instructions for the Little Turtle BioDigital Green Digital PCR instrument to inject the reaction system into the reaction chip. Cycling conditions: 50°C for 10 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 30 seconds, 56°C for 15 seconds, and 72°C for 15 seconds. After cycling, read the signal on the Little Turtle BioDigital Green Chip Analyzer for analysis.

[0250] 1.2 Results

[0251] The results are as follows Figure 2-Figure 4 Primers P1-F / R showed the best results and were selected for use with the probe in subsequent experiments. Primers P2-F / R showed a lower signal, and primers P3-F / R showed double clusters and were excluded.

[0252] Example 2 Design and screening of primers and probes specific for the TERT promoter C228T / C250T mutation

[0253] 2.1 Primer design and screening

[0254] 2.1.1 Materials and methods

[0255] (1) Sample source:

[0256] Positive control: TERT promoter C228T mutant cell line HepG genomic DNA and synthetic C250T mutant plasmid;

[0257] Negative control: genomic DNA from the TERT promoter wild-type cell line 293T.

[0258] (2) Primer design

[0259] Based on the TERT promoter C228T / C250T mutation site and primer design principles, 5 pairs of candidate primers (P4-P8) were designed, as shown in Table 2 below.

[0260] Table 2

[0261]

[0262] (3) Preliminary screening

[0263] Primer screening: Prepare PCR reaction system: 5×HS Taq Buffer with Mg 2+ 4μL, dNTPs (10mM each) 0.5μL, HotStart Taq DNA Polymerase 0.2μL, F primer (10μM) 1μL, R primer (100μM) 1μL, template 2μL, add water to make up to 20μL.

[0264] Cycling conditions: 95°C for 10 min, 40 cycles (95°C for 30 s, 56°C for 15 s, 72°C for 15 s)

[0265] Detection was performed by electrophoresis of 10 μL PCR product.

[0266] 2.1.2 Results

[0267] The results are as follows Figure 5 As shown in the figure, primers P4-F / R produced the correct band but also two nonspecific bands (the top band). Primers P5-F / R produced a weaker amplified band with more nonspecific bands. Primers P6-F / R and P7-F / R produced a single band of incorrect size, and primer P8-F / R produced no clear amplified band. Therefore, primers P4-F / R were selected for subsequent experiments.

[0268] 2.2 Probe design

[0269] 2.2.1 Design Method

[0270] Because C228T / C250T are closely spaced within the same amplicon but do not overlap, and the frequency of simultaneous mutations at both sites is very low, two probes were designed that target the mutant sites but bind to the wild-type template, each labeled with a different fluorophore (see Table 3 below). When the template in the droplet is wild-type, both probes bind and are hydrolyzed simultaneously, resulting in two fluorescent signals in the droplet. When the template in the droplet contains a mutation at one of the sites, the probe at that site cannot bind (e.g., ROX), while the probe at the other site (e.g., Cy5) binds normally and is hydrolyzed, producing a single fluorescent signal (Cy5).

[0271] Table 3

[0272]

[0273] Note: “+T” indicates locked nucleotide T, and “+C” indicates locked nucleotide C.

[0274] Digital PCR experiments:

[0275] Prepare PCR reaction system: 5× HS Taq Buffer with Mg 2+ 6μL, dNTPs (10mM each) 0.75μL, HotStart Taq DNA Polymerase 0.2μL, P4-F / R primers (10μM) 1μL each, C228T / C250T WT probe (10μM) 0.5μL each, template (wild type or mutant) 2μL, add water to make up to 30μL.

[0276] Follow the instructions for the Little Turtle BioDigital Green Digital PCR instrument to inject the reaction system into the reaction chip. Cycling conditions: 50°C for 10 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 30 seconds, 56°C for 15 seconds, and 72°C for 15 seconds. After cycling, read the signal on the Little Turtle BioDigital Green Chip Analyzer for analysis.

[0277] 2.2.2 Results

[0278] The results are as follows Figure 6 As shown, the wild-type template has a weak signal (signal point at a 45° angle upward), is scattered, and has a poor effect.

[0279] Example 3 Optimization of the reaction system for detecting the TERT promoter C228T / C250T mutation

[0280] A PCR reaction system was prepared according to the system described in Example 2, and a final concentration of 1-20% glycerol, 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, was added. Digital PCR detection was performed according to the method in Example 2. It was found that the best signal was obtained when 10% glycerol and 0.5% Poloxamer 188 were added, as shown in FIG. Figure 7-Figure 9 (They are wild-type template, C228T mutant template (red mutation signal point) and C250T mutant template (yellow mutation signal point) in order).

[0281] Example 4 Design and screening of primers for the KRAS G12 / G13 hotspot region

[0282] 4.1 Materials and Methods

[0283] (1) Sample source:

[0284] Positive control: KRAS G12 / G13 hotspot mutant cell lines Sw1116, MM-UC-3, A549, CAL-62, HCT-116, NCL-H1734 genomic DNA and artificially synthesized plasmids;

[0285] Negative control: genomic DNA from the KRAS wild-type cell line 293T.

[0286] (2) Primer design

[0287] Based on the KRAS G12 / G13 hotspot mutation sites and primer probe design principles, 5 pairs of candidate primers (P9-P13) were designed, as shown in Table 4 below.

[0288] Table 4

[0289]

[0290] (3) Primer screening

[0291] A common PCR reaction system was prepared according to Example 2, using primers P9 to P13 as primers, and electrophoresis was performed after the cycle was completed.

[0292] 4.2 Results

[0293] The results are as follows Figure 10 As shown in the figure, primers P9, P10, and P11 can all amplify the correct bands, while primers P12 and P13 cannot amplify the correct bands. Therefore, primer P9 was selected for subsequent experiments.

[0294] Example 5 KRAS G12 / G13 hotspot mutation probe design

[0295] According to the primer and probe design principles screened in Example 4, 9 probes were designed, including one wild-type WT probe that labeled HEX and BHQ1; the other 8 probes targeted 8 mutations, labeled FAM and BHQ1, respectively, as shown in Table 5 below.

[0296] Table 5

[0297]

[0298] Note: “+A” represents locked nucleotide A, “+T” represents locked nucleotide T, “+C” represents locked nucleotide C, and “+G” represents locked nucleotide G.

[0299] According to the above example, digital PCR detection was performed, and the 8 mutation probes were combined with the WT probe to detect the corresponding mutations. The detection results of G12A, G12C, G12D, G12S, G12R, G12V, G13C and G13D were as follows: Figures 11-18 shown.

[0300] Example 6 Detection of 8 mutations in the KRAS G12 / G13 hotspot region using 3 mutation probes

[0301] For thyroid cancer, KRAS mutation detection does not require distinguishing the specific mutation type, so a smaller number of probes was considered to detect eight mutations. After thermodynamic calculations, a combination of four probes (SEQ ID NOs. 31, 34, 36, and 39) was selected for detection (one wild-type probe as an internal reference and three mutant probes to detect mutations).

[0302] The digital PCR detection process is the same as above. The detection results of G12A, G12C, G12D, G12S, G12R, G12V, G13C and G13D are as follows: Figures 19-26 As shown, the combination of these four probes can detect eight mutations.

[0303] Example 7: Simultaneous detection of BRAF V600E, KRAS (G12 / G13 codon hotspot region) and TERT promoter (C228T / C250T) mutations using one reaction

[0304] To simultaneously detect these mutations, KRAS G12 / G13 codon hotspot probes (SEQ ID NOs. 31, 34, 36, and 39) were resynthesized. The fluorescent channel of SEQ ID NO. 31 (wild-type probe) was removed, and the 3' end was phosphorylated to prevent probe extension (C3, C18, or other blocking methods were also possible). The fluorescent moiety of SEQ ID NOs. 34, 36, and 39 was changed to Cy5.5, and the quencher moiety was changed to BHQ3. The BRAF V600E and TERT probes were selected as described in the previous examples.

[0305] The reaction system was prepared as in Example 3, specifically containing 10% glycerol and 0.5% Poloxamer 188. Respective positive and negative controls were tested.

[0306] The results showed that the specificity of the original single-plex digital PCR was still maintained after the combination.

[0307] Example 8 Sensitivity of Multiplex Detection

[0308] A series of positive samples with low mutation ratios were prepared (as shown in Table 6) and tested according to the primer-probe combination of Example 7. Each sample was tested 20 times. The test results of each mutation site are summarized in Table 7 below.

[0309] Table 6

[0310]

[0311] Table 7

[0312]

[0313] The results showed that when the mutation ratio of each mutation site tested was 0.1%, it could meet at least 95% (19 times) positivity, so the minimum detection limit of the detection reagent was determined to be 0.1%.

[0314] discuss

[0315] This invention, based on multiplex digital PCR technology combined with specialized reaction system additives, enables the first simultaneous detection of BRAF V600E (c.1799T>A), KRAS (G12 / G13 codon hotspot), and TERT (C228T / C250T) mutations in thyroid tissue / cytology samples. By optimizing primers, probes, and the reaction system, the assay sensitivity is increased to 0.1% (detecting 5 copies of mutant DNA per reaction system), and the detection cycle is shortened to just 2 hours. Compared to existing technologies, this kit significantly reduces the sample requirement to a single slide (5μm thickness) while maintaining compatibility with paraffin-embedded samples. It is particularly suitable for rapid molecular diagnosis of preoperative FNA specimens.

[0316] This invention overcomes the technical defects of insufficient sensitivity of traditional PCR and complex operation of NGS, providing an efficient and low-cost solution for the precise diagnosis and treatment of thyroid cancer.

[0317] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A reagent for detecting gene mutation, characterized in that: The reagents include: (a) a first primer pair for detecting a BRAF V600E mutation, the first primer pair comprising primers set forth in SEQ ID NOs: 1 and 2; and a first probe for detecting a BRAF V600E mutation used in conjunction with the first primer pair, the first probe being a combination of the following probes: the probe set forth in SEQ ID NO: 7 and the probe set forth in SEQ ID NO: 8; (b) a second primer pair for detecting mutations in the KRAS G12 and G13 hotspot regions, the second primer pair comprising primers set forth in SEQ ID NOs: 21 and 22; and a second probe for detecting mutations in the KRAS G12 and G13 hotspot regions used in conjunction with the second primer pair, the second probe being a combination of the following probes: a probe set forth in SEQ ID NO: 31, a probe set forth in SEQ ID NO: 34, a probe set forth in SEQ ID NO: 36, and a probe set forth in SEQ ID NO: 39; and (c) a third primer pair for detecting TERT promoter C228T mutation and C250T mutation, the third primer pair comprising primers shown in SEQ ID NOs: 9 and 10; and a third probe for detecting TERT promoter C228T mutation and C250T mutation used in conjunction with the third primer pair, the third probe being a combination of the following probes: the probe shown in SEQ ID NO: 19 and the probe shown in SEQ ID NO:

20.

2. The reagent according to claim 1, wherein The BRAF V600E mutation refers to the mutation of valine V at position 600 in the amino acid sequence of the BRAF protein to glutamic acid E; The TERT promoter C228T mutation refers to the mutation of cytosine C at position 228 of the TERT promoter gene nucleotide sequence to thymine T; the TERT promoter C250T mutation refers to the mutation of cytosine C at position 250 of the TERT promoter gene nucleotide sequence to thymine T.

3. The reagent according to claim 1, wherein The KRAS G12 and G13 hotspot mutations include the following mutations: (1) The glycine G at position 12 of the KRAS protein amino acid sequence mutates to alanine A, i.e., G12A; (2) The glycine G at position 12 of the KRAS protein amino acid sequence mutates to cysteine ​​C, i.e., G12C; (3) The glycine G at position 12 of the KRAS protein amino acid sequence mutates to aspartic acid D, i.e., G12D; (4) The glycine G at position 12 of the KRAS protein amino acid sequence mutates to serine S, i.e., G12S; (5) The glycine G at position 12 of the KRAS protein amino acid sequence mutates to arginine R, i.e., G12R; (6) The glycine G at position 12 of the KRAS protein amino acid sequence mutates to valine V, i.e., G12V; (7) The glycine G at position 13 of the KRAS protein amino acid sequence mutates to cysteine ​​C, i.e., G13C; (8) The glycine G at position 13 of the KRAS protein amino acid sequence mutates 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 nucleotide T, "+C" represents locked nucleotide C, and "+A" represents locked nucleotide A.

5. A kit, characterized in that The kit contains the reagent for detecting gene mutation according to claim 1.

6. The kit according to claim 5, wherein The primers and probes in the reagents exist in the form of a primer-probe mixture.

7. The kit according to claim 5, wherein The kit also includes a positive quality control product and a negative quality control product.

8. A method for non-diagnostic in vitro detection of whether a sample contains a gene mutation, characterized in that: Including steps: (s1) providing a PCR reaction system, wherein the PCR reaction system contains a sample to be tested as a template and the reagent for detecting gene mutation according to claim 1; (s2) performing a PCR reaction on the PCR reaction system of step (s1) to obtain an amplified product; (s3) analyzing the amplified product produced in step (s2) to obtain an analysis result of whether the sample to be tested contains a gene mutation.

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