Primer probe combination for verifying whether gene mutation occurs in true gene or not and application of primer probe combination

Abstract

The invention relates to the technical field of molecular biology detection, and discloses a primer probe combination for verifying whether gene mutation occurs in a true gene or not and application of the primer probe combination, and the primer probe combination comprises the following components: a forward primer, a reverse primer and a Blocker. Sequence overlapping exists between the Blocker and the forward primer, and the Blocker is designed to be complementary with the homologous gene and not complementary with the true gene at the difference site of the true gene and the homologous gene, so that the Blocker and the forward primer are competitively combined with the genome DNA, and amplification of the homologous gene is inhibited. And performing Sanger sequencing on the amplification product, comparing mutation sites and difference sites of the true gene and the homologous gene in a sequencing result, and judging whether the gene mutation occurs on the true gene or not. The established method successfully verifies that the prenatal fetal sample SLC6A8 NM005629.4 c.1540 Cgt is used for detecting the prenatal fetal sample SLC6A8 NM005629.4 T (p.R < 514 * >) and SDHA NM004168.4 c.433 Cgt are added into the kit; t (p.Q145 *) mutation authenticity can be realized, and variation sources can be inferred based on family analysis. The method has the advantages of simplicity and convenience in operation, high specificity, low cost, facilitation of popularization and the like.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, specifically to a primer-probe combination for verifying whether gene mutations occur in authentic genes and its application. Background Technology

[0002] Prenatal genetic testing for hereditary diseases is a crucial technology for reducing birth defects and ensuring maternal and infant health, and the accuracy of the results directly determines the rationality of clinical decisions. Homologous genes, as genomic segments highly similar to functional eukaryotic sequences but without coding function, constitute a very large proportion of the human genome (approximately 13,343), and their presence can easily interfere with the interpretation of genetic testing results. If it is impossible to accurately distinguish whether pathogenic mutations occur in eukaryotic or homologous genes, it may lead to incorrect mutation identification, resulting in misdiagnosis or missed diagnosis, posing a serious threat to the reliability of prenatal diagnosis. Therefore, establishing accurate methods for identifying eukaryotic and homologous gene mutations is a core requirement for improving the accuracy of prenatal diagnosis of hereditary diseases and has significant clinical implications.

[0003] In existing technologies, the identification methods for eugenic and homologous gene mutations mainly revolve around sequencing technology optimization and specific amplification strategies: one is to rely on sequencing technology improvements, such as extending read lengths and optimizing transcript splicing algorithms, in an attempt to reduce splicing errors caused by sequence similarity in short-read NGS technology, thereby indirectly reducing homologous gene interference; the other is to usually rely on complex PCR, such as nested PCR or long-range-PCR (LR-PCR), and then perform NGS sequencing on the PCR products to achieve the identification of homologous genes.

[0004] However, existing technologies still have the following technical problems: (1) Existing sequencing technologies have limited optimization effects. Even with improved splicing algorithms, short-read NGS still cannot completely avoid interference from highly homologous gene sequences, and cannot fundamentally solve the problem of distinguishing between eugenic and homologous gene variations; (2) Existing primer designs have limitations. Eugenic and homologous gene sequences are highly similar with few differential sites. Traditional specific primers are prone to non-specific binding, resulting in low identification accuracy; (3) Existing third-generation sequencing and complex PCR amplification both have disadvantages such as long development cycles and insufficient flexibility, which cannot meet the stringent requirements of prenatal diagnosis for result reliability. In summary, existing methods are difficult to accurately and efficiently distinguish between eugenic and homologous gene mutations. There is an urgent need for a highly specific and accurate identification scheme to solve the problem of homologous gene interference in prenatal gene testing. Summary of the Invention

[0005] The present invention aims to provide a primer-probe combination for verifying whether gene mutations occur in primordial genes and its application, in order to solve the technical problem that existing methods are difficult to accurately and efficiently distinguish between primordial genes and homologous gene mutations.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a primer-probe combination for verifying whether a gene mutation occurs in a virgin gene, comprising a Blocker sequence, a forward primer, and a reverse primer; the Blocker sequence is designed at one of the differential sites of the virgin gene and the homologous gene near the mutation site to be verified, the Blocker sequence partially overlaps with the forward primer, is complementary to the homologous gene, is not complementary to the virgin gene, and has a 5-6 nt overhang at the 3' end, to avoid non-specific amplification caused by the Blocker.

[0007] Preferably, as an improvement, the concentration of the Blocker sequence is 10 times that of the concentrations of the forward and reverse primers. This is used to bind homologous genes, effectively inhibiting homologous gene amplification, thereby achieving specific amplification of the true gene.

[0008] Preferably, as an improvement, the length of the Blocker sequence is 28~35nt, and the overlap between the Blocker and the forward primer bases is 6~10nt.

[0009] Preferably, as an improvement, the sequence lengths of the forward and reverse primers are 18-24 nt, and the difference in Tm values ​​between the forward and reverse primers is less than 5℃.

[0010] Preferably, as an improvement, when the true gene containing the mutation site to be verified is the SLC6A8 gene and the mutation to be detected is NM_005629.4 c.1540 C>T (p.R514*), the primer-probe combination includes SLC6A8-FP, SLC6A8-Blocker, and SLC6A8-RP, and their nucleotide sequences are shown in SEQ_ID_NO.1, SEQ_ID_NO.2, and SEQ_ID_NO.3, respectively.

[0011] The mutation “NM_005629.4c.1540C>T(p.R514*)” means that “in the specific reference sequence version NM_005629.4 of the SLC6A8 gene, nucleotide 1540 of the DNA sequence has undergone a C (cytosine) to T (thymine) change. This change results in the encoding of amino acid 514, which was normally arginine, becoming a stop codon.” This typically means that protein synthesis is prematurely interrupted, resulting in a truncated and nonfunctional protein. This type of mutation is classified as a “Class 1 – Pathogenic Mutation” according to the ACMG guidelines.

[0012] Preferably, as an improvement, the nucleotide sequence of the wild-type SLC6A8 gene is shown in SEQ_ID_NO.4, the nucleotide sequence of the mutant SLC6A8 gene is shown in SEQ_ID_NO.5, and the nucleotide sequence of the homolog of the SLC6A8 gene, SLC6A10P, is shown in SEQ_ID_NO.6.

[0013] Preferably, as an improvement, when the true gene containing the mutation site to be verified is the SDHA gene and the mutation to be detected is NM_004168.4 c.433 C>T(p.Q145*), the primer-probe combination includes SDHA-FP, SDHA-Blocker, and SDHA-RP, and their nucleotide sequences are shown in SEQ_ID_NO.7, SEQ_ID_NO.8, and SEQ_ID_NO.9, respectively.

[0014] The mutation “NM_004168.4 c.433 C>T(p.Q145*)” means that “in the NM_004168.4 reference sequence version of the SDHA gene, nucleotide 433 of the DNA sequence has undergone a C (cytosine) to T (thymine) change. This change results in amino acid 145 being replaced by a stop codon instead of the normal glutamine.” This is a typical nonsense mutation that leads to premature termination of protein synthesis. According to the ACMG guidelines, this variant is classified as “Class 2 – Probably pathogenic”.

[0015] Preferably, as an improvement, the nucleotide sequence of the wild-type SDHA gene is shown in SEQ_ID_NO.10, the nucleotide sequence of the mutant SDHA gene is shown in SEQ_ID_NO.11, the nucleotide sequence of the homologous gene SDHAP2 of the SDHA gene is shown in SEQ_ID_NO.12, and the nucleotide sequence of the homologous gene SDHAP4 of the SDHA gene is shown in SEQ_ID_NO.13.

[0016] Preferably, as an improvement, this solution also provides an application of primer-probe combination in the verification of point mutation origin, including using the above primer-probe combination to verify whether gene mutation occurs in a true gene.

[0017] Preferably, as an improvement, the verification approach is as follows: The amplified product is used for Sanger sequencing, and the Sanger sequencing results are analyzed to interpret the gene origin and pedigree transmission pattern of the point mutation: When the sequencing result is a single peak, and the differentially expressed site on the sequence containing the mutation matches the virgin gene, the mutation occurs in the virgin gene; otherwise, the possibility that the mutation occurs in a homologous gene needs to be considered. The Blocker sequence needs to be redesigned so that it is completely complementary to the virgin gene at the differentially expressed sites of the covered virgin and homologous genes, but not complementary to homologous genes, specifically enriching homologous genes. The source of the mutation is then determined by combining this with the Sanger sequencing results.

[0018] The principles and advantages of this solution: This protocol develops a primer-probe combination to inhibit homologous gene amplification, and then utilizes a blocker complementary to the homologous gene to further suppress homologous gene amplification, achieving the goal of specifically enriching the genotype. The amplified products are then subjected to Sanger sequencing, and the sequencing results are compared with the genotype and homologous gene sequences to determine the authenticity of the mutation. Specifically, in this primer-probe combination, differential sites between the genotype and homologous gene are identified within a 50-500 bp range upstream and downstream of the mutation site to be verified. A blocker is designed based on one of these differential sites, ensuring that the blocker is complementary to the homologous gene but not to the genotype, and that it has a 5-6 nt overhang at its 3' end to prevent non-specific amplification caused by blocker extension. The forward and reverse primers are common primers, and the blocker is designed to partially overlap with the forward primer. When the amplification reaction is in progress, the forward primer that binds to the homologous gene is blocked by the blocker, thus inhibiting the amplification of the homologous gene; since there is no complementarity between the blocker and the true gene, it is easily replaced by the forward primer during amplification, the amplification proceeds smoothly, the true gene is enriched, and the specificity of amplification is improved.

[0019] In practical applications, the amplified products are used for Sanger sequencing. Analysis of the Sanger sequencing results helps interpret the gene origin and familial transmission pattern of point mutations. When the sequencing result is a single peak, and the differentially expressed site on the mutation site matches the true gene, the mutation occurs in the true gene. In familial samples, if the mother and fetus have the same mutation at the same site of the same gene, and the father has no mutation, the fetal genomic DNA mutation originates from the mother; if the father and fetus have the same mutation at the same site of the same gene, and the mother has no mutation, the fetal genomic DNA mutation originates from the father; if only the fetus has the mutation, and neither the father nor the mother has the mutation, the fetal genomic DNA mutation is a de novo mutation. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the primer-probe combination design principle provided in the embodiments of the present invention.

[0021] Figure 2 This is the amplification curve of the SLC6A8 gene c.1540 C>T in Example 1 of the present invention.

[0022] Figure 3 This is the melting curve of the SLC6A8 gene c.1540 C>T amplified in Example 1 of this invention.

[0023] Figure 4 This is a schematic diagram of the sequence of the true gene SLC6A8 and its homolog SLC6A10P in Example 1 of the present invention.

[0024] Figure 5 This is a sequencing result diagram of the SLC6A8 gene c.1540 C>T in the fetus of family (I) in Example 1 of the present invention.

[0025] Figure 6 This is a sequencing result diagram of the SLC6A8 gene c.1540 C>T detected in the mother of family (I) in Example 1 of the present invention.

[0026] Figure 7 This is a sequencing result diagram of the SLC6A8 gene c.1540 C>T detected in the father of family (I) in Example 1 of the present invention.

[0027] Figure 8 This is a schematic diagram of the sequence of the true gene SDHA and its homologous genes SDHAP2 and SDHAP4 in Embodiment 2 of the present invention.

[0028] Figure 9 This is a sequencing result diagram of the SDHA gene c.433 C>T in the fetus of family (II) in Example 2 of the present invention.

[0029] Figure 10 This is a sequencing result diagram of the SDHA gene c.433 C>T detected in the mother of family (II) in Example 2 of the present invention.

[0030] Figure 11 This is a sequencing result diagram of the SDHA gene c.433 C>T detected in the father of family (II) in Example 2 of the present invention. Detailed Implementation The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used are all commercially available.

[0031] Example 1 This protocol provides a primer-probe combination for verifying whether a gene mutation occurs in a primordial gene, including a Blocker sequence, a forward primer, and a reverse primer. The Blocker sequence is designed at one of the differential sites between the primordial gene and a homologous gene near the mutation site to be verified. The Blocker sequence partially overlaps with the forward primer, is complementary to the homologous gene, is not complementary to the primordial gene, and has a 5-6 nt overhang at the 3' end.

[0032] As an improvement, the length of the Blocker sequence is 28~35nt, and the overlap between the Blocker and the forward primer bases is 6~10nt.

[0033] The sequence lengths of the forward and reverse primers are 18–24 nt, and the difference in Tm values ​​between the forward and reverse primers is less than 5 °C.

[0034] When the true gene containing the mutation site to be verified is the SLC6A8 gene and the mutation to be detected is NM_005629.4c.1540 C>T (p.R514*), the primer-probe combination includes SLC6A8-FP, SLC6A8-Blocker, and SLC6A8-RP, and their nucleotide sequences are shown in SEQ_ID_NO.1, SEQ_ID_NO.2, and SEQ_ID_NO.3, respectively.

[0035] The nucleotide sequence of the wild-type SLC6A8 gene is shown in SEQ_ID_NO.4, the nucleotide sequence of the mutant SLC6A8 gene is shown in SEQ_ID_NO.5, and the nucleotide sequence of the homolog of SLC6A8, SLC6A10P (i.e., the homolog SLC6A10P), is shown in SEQ_ID_NO.6.

[0036] When the true gene containing the mutation site to be verified is the SDHA gene and the mutation to be detected is NM_004168.4 c.433C>T(p.Q145*), the primer-probe combination includes SDHA-FP, SDHA-Blocker, and SDHA-RP, and their nucleotide sequences are shown in SEQ_ID_NO.7, SEQ_ID_NO.8, and SEQ_ID_NO.9, respectively.

[0037] The nucleotide sequence of the wild-type SDHA gene is shown in SEQ_ID_NO.10, the nucleotide sequence of the mutant SDHA gene is shown in SEQ_ID_NO.11, the nucleotide sequence of the homologous gene SDHAP2 of SDHA is shown in SEQ_ID_NO.12, and the nucleotide sequence of the homologous gene SDHAP4 of SDHA is shown in SEQ_ID_NO.13.

[0038] This solution also provides a method for designing primer-probe combinations to verify whether gene mutations occur in primordial genes, such as... Figure 1 As shown, the process involves designing a Blocker sequence at one of the differential sites between the genogene and the homologous gene near the mutation site to be verified, and then designing forward and reverse primers. The forward and reverse primers are common primers. The Blocker sequence partially overlaps with the forward primer, is complementary to the homologous gene, is not complementary to the genogene, and has a 5-6 nt overhang at the 3' end.

[0039] As a reference, the length of the Blocker sequence is 28~35nt, and the overlap between the Blocker and the forward primer is 6~10nt; while the length of the forward and reverse primer sequences is 18~24nt, and the difference in Tm values ​​between the forward and reverse primers is less than 5℃.

[0040] When preparing products using the primer-probe combination of this scheme, the concentration of the Blocker sequence is 10 times that of the forward and reverse primers.

[0041] This protocol also provides an application of primer-probe combinations in verifying the origin of point mutations, including using the aforementioned primer-probe combinations to verify whether gene mutations occur in primordial genes. The verification approach is as follows: the amplified products are used for Sanger sequencing, and the Sanger sequencing results are analyzed to interpret the gene origin and pedigree transmission pattern of the point mutation: when the sequencing result is a single peak, and the differentially expressed site on the mutation site matches the primordial gene, the mutation occurs in the primordial gene; otherwise, the possibility of the mutation occurring in a homologous gene needs to be considered. The Blocker sequence needs to be redesigned so that it is completely complementary to the primordial gene at the differentially expressed sites of the covered primordial and homologous genes, while exhibiting non-complementarity with homologous genes, specifically enriching homologous genes. The mutation origin is then determined in conjunction with the Sanger sequencing results.

[0042] Example 1 This embodiment provides a primer-probe combination for verifying whether the SLC6A8 gene NM_005629.4 c.1540 C>T (p.R514*) occurs in a true gene, and verifies the fetal mutation origin in clinical prenatal family samples. The primer-probe combination includes: SLC6A8-FP, SLC6A8-Blocker, and SLC6A8-RP.

[0043] Specifically, the primer and probe sequences, the covered eugenic and homologous gene sequences are shown in Table 1 below.

[0044] Table 1. Primer and probe sequences related to the SLC6A8 gene, the covered eukaryotic and homologous gene sequences.

[0045] Note: In FP and Blocker, the bold black "AGGAGCTGACC" represents the overlapping sequence between the Blocker and the forward primer; in the Blocker sequence, the underlined italicized bases " atata "T" represents the dangling sequence at the 3' end of the Blocker that is not complementary to the genomic DNA; in mutant sequences, a single bold black base "T" represents the gene mutation site; in homologous gene sequences, single bold black bases "C" and "T" represent the differential sites between the SLC6A8 gene and its homolog (SLC6A10P).

[0046] The reaction system was prepared as shown in Table 2.

[0047] Table 2 Reaction System

[0048] Add 3 μL of genomic DNA to the prepared amplification reaction system.

[0049] After adding and mixing the samples, the amplification reaction was performed using an ABI 7500 real-time quantitative PCR instrument. The reaction procedure is as follows: First stage: React at 95℃ for 5 minutes; Second stage: react at 95℃ for 10s, react at 58℃ for 30s, repeat 60 times, and collect fluorescence signals; Phase 3: Melt (95℃~60℃); The amplification curve obtained from the amplification reaction is as follows: Figure 2 As shown, the melting curve is as follows Figure 3 As shown, the Sanger sequencing results of the amplified products are as follows: Figures 5-7 As shown.

[0050] The results show: In this embodiment, the melting curves of the amplification products from the family samples obtained were all single-peaked, and the sequencing results of the products were also all single-peaked, indicating that the amplification products were homogeneous. Differential sites of eugenics and homologous genes were used as references (e.g., ...). Figure 4As shown in the diagram, it can be determined that the SLC6A8 gene NM_005629.4 c.1540 C>T(p.R514*) in both the fetus and mother is a true genotype, and the father of the fetus did not have the mutation. SLC6A8 gene-related diseases include cerebral creatine deficiency syndrome type 1, with an X-linked inheritance pattern. Patients are typically male, and clinical manifestations mainly include intellectual disability, severe language delay, behavioral abnormalities, and epilepsy. Heterozygous female carriers may also exhibit clinical manifestations, primarily mild intellectual disability, learning difficulties, and constipation. The fetus is male, and based on family pedigree analysis, the fetus's SLC6A8 gene NM_005629.4 c.1540 C>T(p.R514*) is hemizygous, originating from the mother, and this result is consistent with the next-generation sequencing results.

[0051] Example 2 This embodiment provides a primer-probe combination for verifying whether the SDHA gene NM_004168.4 c.433 C>T(p.Q145*) occurs in a true gene, and verifies the fetal mutation origin in clinical prenatal family samples. The primer-probe combination includes: SDHA-FP, SDHA-Blocker, and SDHA-RP.

[0052] Specifically, the primer and probe sequences, the covered eugenic and homologous gene sequences are shown in Table 3 below.

[0053] Table 3 Primer and probe sequences of the SDHA gene, covering eukaryotic and homologous gene sequences.

[0054] Note: The bold "TTTCCAGG" in FP and Blocker indicates the overlapping sequence between the Blocker and the forward primer; the underlined italic "TTTCCAGG" in the Blocker sequence indicates the overlapping sequence between the Blocker and the forward primer. aaaaa "" represents a hanging sequence at the 3' end of the Blocker that is not complementary to the genomic DNA; the bold black "T" in the mutant sequence represents the gene mutation site; in two homologous genes Black bold and underlined The bases represent the different sites between the SDHA gene and its homologs (SDHAP2, SDHAP4).

[0055] The reaction system was prepared as shown in Table 4.

[0056] Table 4 Reaction System

[0057] Add 3 μL of genomic DNA to the prepared amplification reaction system.

[0058] After adding and mixing the samples, the amplification reaction was performed using an ABI 7500 real-time quantitative PCR instrument. The reaction procedure is as follows: First stage: React at 95℃ for 5 minutes; Second stage: react at 95℃ for 10s, react at 58℃ for 30s, repeat 60 times, and collect fluorescence signals; Phase 3: Melt (95℃ ~ 60℃); Sanger sequencing results of the amplified products are as follows Figures 8-11 As shown.

[0059] The results show: The Sanger sequencing results of the amplified products from the family samples obtained in Example 2 all showed single peaks, which can be used for result interpretation. Differential sites in eugenics and homologous genes were used as references (e.g., ...). Figure 8 As shown in the diagram, it can be determined that the SDHA gene NM_004168.4 c.433 C>T(p.Q145*) in both the fetus and the father is a primordial mutation, and no mutation was found in the fetus's mother. Combined with family pedigree analysis, it can be determined that the fetus's SDHA gene NM_004168.4 c.433 C>T(p.Q145*) originates from the father, and this result is consistent with the next-generation sequencing results.

[0060] The method provided by this invention requires only one pair of amplification primers and one unmodified blocker to specifically enrich true genes and inhibit the amplification of homologous genes. The amplification products can be Sanger sequenced and compared with reference genome sequences and homologous genes to determine the authenticity of mutations. Furthermore, the source of variation can be determined based on family analysis. This method has advantages such as simple operation, high specificity, low cost, and ease of widespread adoption. The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A primer-probe combination for verifying whether a gene mutation occurs in a virgin gene, characterized in that: It includes a Blocker sequence, a forward primer, and a reverse primer; the Blocker sequence is designed at one of the differential sites of the eugenic gene and the homologous gene near the mutation site to be verified. The Blocker sequence partially overlaps with the forward primer, is complementary to the homologous gene, is not complementary to the eugenic gene, and has a 5-6 nt overhang at the 3' end.

2. The primer-probe combination according to claim 1, characterized in that: The concentration of the Blocker sequence is 10 times that of the concentrations of the forward and reverse primers.

3. The primer-probe combination according to claim 1, characterized in that: The length of the Blocker sequence is 28-35 nt, and the overlap between the Blocker and the forward primer bases is 6-10 nt.

4. The primer-probe combination according to claim 1, characterized in that: The sequence lengths of the forward and reverse primers are 18-24 nt, and the difference in Tm values ​​between the forward and reverse primers is less than 5℃.

5. The primer-probe combination according to claim 1, characterized in that: When the gene containing the mutation site to be verified is the SLC6A8 gene and the mutation to be detected is NM_005629.4 c.1540 C>T (p.R514*), the primer-probe combination includes SLC6A8-FP, SLC6A8-Blocker, and SLC6A8-RP, and their nucleotide sequences are shown in SEQ_ID_NO.1, SEQ_ID_NO.2, and SEQ_ID_NO.3, respectively.

6. The primer-probe combination according to claim 5, characterized in that: The nucleotide sequence of the wild-type SLC6A8 gene is shown in SEQ_ID_NO.4, the nucleotide sequence of the mutant SLC6A8 gene is shown in SEQ_ID_NO.5, and the nucleotide sequence of the homolog of the SLC6A8 gene, SLC6A10P, is shown in SEQ_ID_NO.

6.

7. The primer-probe combination according to claim 1, characterized in that: When the true gene containing the mutation site to be verified is the SDHA gene and the mutation to be detected is NM_004168.4 c.433 C>T(p.Q145*), the primer-probe combination includes SDHA-FP, SDHA-Blocker, and SDHA-RP, and their nucleotide sequences are shown in SEQ_ID_NO.7, SEQ_ID_NO.8, and SEQ_ID_NO.9, respectively.

8. The primer-probe combination according to claim 7, characterized in that: The nucleotide sequence of the wild-type SDHA gene is shown in SEQ_ID_NO.10, the nucleotide sequence of the mutant SDHA gene is shown in SEQ_ID_NO.11, the nucleotide sequence of the homologous gene SDHAP2 of the SDHA gene is shown in SEQ_ID_NO.12, and the nucleotide sequence of the homologous gene SDHAP4 of the SDHA gene is shown in SEQ_ID_NO.

13.

9. An application of a primer-probe combination in verifying the origin of point mutations, characterized in that: This includes using the primer-probe combination according to any one of claims 1 to 8 to verify whether a gene mutation occurs in a true gene.

10. The application according to claim 9, characterized in that: The verification approach is as follows: the amplified products are used for Sanger sequencing, the Sanger sequencing results are analyzed, and the gene origin and pedigree transmission pattern of point mutations are interpreted: when the sequencing result is a single peak, and the differentially expressed site on the sequence where the mutation site is located matches the true gene, the mutation occurs on the true gene.

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