CN116814772A - Primer combination, kit and method for detecting FANCA gene mutation - Google Patents

Abstract

The present application relates to a primer combination, kit and method for detecting FANCA gene mutations. The primer combination is selected from primer pair 1 to primer pair 105 with nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 210. Many pairs in. This application provides a highly versatile and accurate method for preimplantation genetic detection of FANCA mutations, which can be used for high-throughput sequencing to detect FANCA mutations at the embryonic level, helping families with children with Fanconi anemia who have a reproductive history. Produce healthy offspring.

Description

Primer combination, kit and method for detecting FANCA gene mutation

Technical Field

The present application relates to the field of molecular biology technology, and in particular to a primer combination, a kit and a method for detecting FANCA gene mutation.

Background Art

Fanconi anemia (FA) is a rare monogenic genetic disease characterized by progressive bone marrow failure and is the most common congenital hematopoietic failure disease. Fanconi anemia can be caused by mutations in the FANCA gene and is inherited in an autosomal recessive manner. It is a disease that affects multiple organs in the body and has high clinical heterogeneity. The main clinical manifestations are physical abnormalities, bone marrow failure, and an increased risk of malignant tumors.

Preimplantation genetic testing (PGT) is an effective means to prevent birth defects such as Fanconi anemia. PGT refers to the process of biopsy of several cells for genetic testing when the embryo develops in vitro to the cleavage stage or blastocyst stage, and finally selects embryos without disease risk to be implanted in the mother's uterus, so as to achieve the purpose of giving birth to healthy offspring. PGT can effectively avoid the physical and mental harm caused by termination of pregnancy due to pregnancy with a fetus with genetic disease, and is increasingly becoming the first choice for couples with a high risk of giving birth to a fetus with genetic disease. Common methods for FANCA gene testing at the embryo level include multiple nested PCR and Karyomapping technology. The multiple nested PCR method requires the pre-screening of heterozygous short tandem repeats (STRs) for FANCA gene carriers, and then establishes a multiple PCR system containing mutation sites and heterozygous STR sites at the lymphocyte level, and evaluates the amplification efficiency and allele dropout rate of each site at the cell level. Only qualified ones can be used for embryo testing. Due to the limited number of STRs and the different heterozygous frequencies in the population, the multiple nested PCR method is more personalized in design, with a large workload, a long cycle, and low versatility. Compared with STR loci, single nucleotide polymorphism loci (SNP) are more numerous and widely distributed, with an occurrence frequency of more than 1% in the population, a total of 3 million, an average of 1/3Kb, and easy to automate analysis on high-throughput sequencing platforms. Karyomapping technology indirectly excludes genetic defects through SNP linkage analysis. There are 300,000 SNP probes on the chip for whole-genome linkage analysis, which is a comprehensive and universal PGT technology for single-gene diseases. However, Karyomapping cannot detect gene mutations, so there must be a proband sample or a suitable family member sample. If there are not enough family samples, additional mutation detection is required.

Therefore, it is urgent to develop a method that can more comprehensively cover the SNP sites within the FANCA gene and its upstream and downstream to determine the FANCA mutation genotype of the embryo.

Summary of the invention

Based on this, it is necessary to provide a primer combination, a kit and a method for detecting FANCA gene mutations with strong versatility and high diagnostic rate.

In a first aspect of the present application, a primer combination for detecting FANCA gene mutation is provided, wherein the primer combination is selected from a plurality of pairs of primer pairs 1 to 105 having nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 210.

In one embodiment, the primer combination includes primer pair 1 to primer pair 105 having nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 210.

The second aspect of the present application provides a kit for detecting FANCA gene mutation, comprising the primer combination.

In one embodiment, the kit also includes PCR reaction reagents.

In one embodiment, the PCR reaction reagents include one or more of PCR buffer, DNA polymerase, Mg2+ and dNTPs.

The third aspect of the present application provides a method for detecting a FANCA gene mutation, comprising the following steps:

Obtain genomic DNA from samples;

The primer combination or the kit is used to perform PCR amplification on the FANCA gene coding region and the upstream and downstream SNP sites of the genomic DNA of the sample; the PCR amplification product is analyzed, and the FANCA mutation type is determined according to the analysis result.

In one embodiment, the FANCA gene coding region and its upstream and downstream SNP sites include: chr16:86817040, chr16:86867197, chr16:86932609, chr16:86970454, chr16:86970479, chr16:86970503, chr16:87019166, chr16:87060416, chr16:87060483, chr16:87111668, chr16:87232383, c hr16:87305229、chr16:87343729、chr16:87402133、chr16:87446368、chr16:87696272、chr16:87714622、chr16:87720579、chr16:87722157、chr16:87727 927, chr16:87730041, chr16:87738853, chr16:87742056, chr16:87748198, chr16:8 7748298、chr16:87748315、chr16:87755154、chr16:87755155、chr16:87756047、chr16:87980060、chr16:87980067、chr16:88038557、chr16:88080922、chr 16:88081884, chr16:88081904, chr16:88081951, chr16:88085428, chr16:8808947 8. chr16:88100321, chr16:88102208, chr16:88106046, chr16:88116559, chr16:88164860, chr16:88164924, chr16:88165210, chr16:88165235, chr16:88 453572, chr16:88460235, chr16:88469873, chr16:88504850, chr16:88520452, chr16 ch r16:88902183, chr16:88902277, chr16:88909028, chr16:88921022, chr16:89042 334. chr16:89161684, chr16:89216941, chr16:89304949, chr16:89437560, chr16:89568875, chr16:89590758, chr16:89600177, chr16:89649245, chr16: 89675319、chr16:89697891、chr16:89800058、chr16:89818732、chr16:89909429、ch r16:89933420、chr16:89950299、chr16:89954138、chr16:89957635、chr16:89958538、chr16:89961597、chr16:89967217、chr16:89972532、chr16:899756 38. chr16:89985222, chr16:89986154, chr16:89994413, chr16:90008296, chr16:90 008896、chr16:90013420、chr16:90020346、chr16:90024206、chr16:90024899、chr16:90031427、chr16:90044028、chr16:90046121、chr16:90060281、chr 16:90074001, chr16:90076227, chr16:90098466, chr16:90098616 and chr16:90107377.

In one embodiment, the method for analyzing the PCR amplification product includes: performing high-throughput sequencing on the PCR amplification product.

In one embodiment, the sample is selected from one or more of peripheral blood, semen, oral mucosal cells and embryonic cells.

The fourth aspect of the present application provides use of the primer combination or the kit in preparing a product for detecting FANCA gene mutation.

Compared with the traditional technology, the present invention also has the following beneficial effects:

The present application provides a primer combination for detecting FANCA gene mutations. By performing PCR amplification on the FANCA gene coding region and its upstream and downstream SNP sites of the sample, the haplotype and genotype of the FANCA of the sample can be detected. The present application uses the above primer combination to construct a method for preimplantation genetic testing with strong versatility and high diagnostic rate, which eliminates the need for preliminary experiments at the cell level and can be used for high-throughput sequencing to detect FANCA mutations at the embryo level, helping families with a history of giving birth to children with Fanconi anemia to give birth to healthy offspring. The advantages of the present application mainly include the following points:

(1) A large number of SNPs detected: The present application can more comprehensively detect the SNP sites in the FANCA gene coding region and its upstream and downstream regions, and can also detect unknown SNP mutation sites.

(2) High versatility and accuracy: The test was conducted on 16 samples, and the passing standard was set as no less than 2 valid SNPs on each side of the gene, and the sample detection pass rate was 100%.

(3) Low cost: When the detection method of the present application is used for detection, the cost of haplotype analysis of FANCA is low.

DETAILED DESCRIPTION

For ease of understanding of the present application, the present application will be described more fully below with reference to the relevant embodiments. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

the term

Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

In the present application, the selection range involving "and/or", "or/and", and "and/or" includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and/or", "or/and", and "and/or", it should be understood that the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or".

In this application, "optionally", "optional" and "optional" refer to optional, that is, to any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

In the present application, the terms “preferred”, “better”, “more preferred” and “suitable” are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of the present application.

In the present application, the terms “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

In this application, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is an integer selected from any one of the integer groups consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and/or" used herein includes any and all combinations of one or more related listed items.

Reads refer to the sequence fragments obtained by sequencing.

Single nucleotide polymorphism (SNP) refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level.

Haplotype, also known as haplotype or unit type, refers to a group of interrelated single nucleotide polymorphisms located in a specific region of a chromosome that tend to be inherited as a whole to offspring.

Sequencing depth refers to the ratio of the total number of bases obtained by sequencing to the size of the genome to be tested, which can be understood as the average number of times each tested base in the genome is repeatedly sequenced (in units of base numbers). In one embodiment of the present application, the sequencing depth is 1000x, which means that the specific PCR amplification product is sequenced 1000 times.

In order to solve the above problems, the first aspect of the present application provides a primer combination for detecting FANCA gene mutation, wherein the primer combination is selected from multiple pairs of primer pairs 1 to primer pairs 105 with nucleotide sequences as shown in SEQ ID No.1 to SEQ ID No.210.

Optionally, the primer combination includes primer pair 1 to primer pair 105 having nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 210.

The second aspect of the present application provides a kit for detecting FANCA gene mutation, comprising the primer combination.

Optionally, the kit further comprises PCR reaction reagents.

Optionally, the PCR reaction reagents include one or more of PCR buffer, DNA polymerase, Mg2+ and dNTPs.

The third aspect of the present application provides a method for detecting a FANCA gene mutation, comprising the following steps:

Obtain genomic DNA from samples;

The primer combination or the kit is used to perform PCR amplification on the FANCA gene coding region and the upstream and downstream SNP sites of the genomic DNA of the sample; the PCR amplification product is analyzed, and the FANCA mutation type is determined according to the analysis result.

Optionally, the FANCA gene coding region and its upstream and downstream SNP sites include: chr16:86817040, chr16:86867197, chr16:86932609, chr16:86970454, chr16:86970479, chr16:86970503, chr16:87019166, chr16:87060416, chr16:87060483, chr16:87111668, chr16:87232383, chr16: 87305229、chr16:87343729、chr16:87402133、chr16:87446368、chr16:87696272、chr16:87714622、chr16:87720579、chr16:87722157、chr16:87727927、 chr16:87730041、chr16:87738853、chr16:87742056、chr16:87748198、chr16:877482 98. chr16:87748315, chr16:87755154, chr16:87755155, chr16:87756047, chr16:87980060, chr16:87980067, chr16:88038557, chr16:88080922, chr16:8 8081884, chr16:88081904, chr16:88081951, chr16:88085428, chr16:88089478, ch r16:88100321, chr16:88102208, chr16:88106046, chr16:88116559, chr16:88164860, chr16:88164924, chr16:88165210, chr16:88165235, chr16:884535 72. chr16:88460235, chr16:88469873, chr16:88504850, chr16:88520452, chr16:88 535620、chr16:88627084、chr16:88635395、chr16:88664758、chr16:88668051、chr16:88680855、chr16:88791441、chr16:88822782、chr16:88880844、chr 16:88902183, chr16:88902277, chr16:88909028, chr16:88921022, chr16:8904233 4. chr16:89161684, chr16:89216941, chr16:89304949, chr16:89437560, chr16:89568875, chr16:89590758, chr16:89600177, chr16:89649245, chr16:89 675319、chr16:89697891、chr16:89800058、chr16:89818732、chr16:89909429、chr1 6:89933420、chr16:89950299、chr16:89954138、chr16:89957635、chr16:89958538、chr16:89961597、chr16:89967217、chr16:89972532、chr16:8997563 8. chr16:89985222, chr16:89986154, chr16:89994413, chr16:90008296, chr16:900 08896, chr16:90013420, chr16:90020346, chr16:90024206, chr16:90024899, chr16:90031427, chr16:90044028, chr16:90046121, chr16:90060281, chr1 6:90074001, chr16:90076227, chr16:90098466, chr16:90098616 and chr16:90107377.

Optionally, the method for analyzing the PCR amplification product includes: performing high-throughput sequencing on the PCR amplification product.

Optionally, the high-throughput sequencing platform is Illumina Miseq.

Optionally, the PCR amplification method is multiplex PCR amplification.

Optionally, the sample is selected from one or more of peripheral blood, semen, oral mucosal cells and embryonic cells.

Further optionally, the sample is embryonic cells at the cleavage stage or blastocyst stage.

Preferably, the DNA content in each DNA sample is greater than 500 ng.

In a specific example, performing high-throughput sequencing on the PCR amplification product includes: comparing the sequencing result with the DNA sequences of both parents of the sample, and analyzing the haplotype of the embryonic cell FANCA.

In a specific example, performing high-throughput sequencing on the PCR amplification product further includes: comparing the sequencing result with the human genome reference sequence, and analyzing the SNP coverage multiples and genotype.

In one specific embodiment, the detection method of the present application is to take a number of cells for genetic testing when the fertilized egg develops in vitro to the cleavage stage or blastocyst stage. It can be understood that this method is not directly implemented on a living human or animal body and does not belong to a method for diagnosing and treating diseases.

The fourth aspect of the present application provides use of the primer combination or the kit in preparing a product for detecting FANCA gene mutation.

The following is further described in conjunction with specific embodiments. The raw materials involved in the following specific embodiments, unless otherwise specified, can all be commercially available. The instruments used, unless otherwise specified, can all be commercially available. The processes involved, unless otherwise specified, are all routinely selected by those skilled in the art.

Example 1

Screening of SNP sites and primer design

1. SNP site screening principles: high-frequency SNPs were selected from the 1000 Genomes Project database (http://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/); high-frequency SNP sites with the lowest allele frequency greater than 0.2; SNP sites with a GC content of >70% in the polynucleotide (polyN) and 50bp sequences upstream and downstream of the site were removed; SNP sites with multiple positions in the upstream and downstream 50bp sequences aligned to the human genome hg19 were removed (i.e., SNP sites with high homology were removed);

SNP selection range: Priority is given to SNP sites within the FANCA gene and within 1Mb upstream and downstream of the gene (or mutation site). If the number of SNPs within 1Mb is small, the range can be appropriately expanded, such as 2Mb upstream and downstream.

According to the above method, the SNP sites in the coding region of the FANCA gene and its upstream and downstream were screened.

2. Primer design: The corresponding primers were designed according to the screening principle of the above SNP sites through an online platform (https://www.ampliseq.com/); the primer sequences are shown in SEQ ID NO.1 to SEQ ID NO.210. The primers have high specificity, similar annealing temperatures and PCR product fragment sizes in the range of 125bp-375bp.

The sequences of the SNP sites and the primers are detailed in Table 1, where sequence numbers 1-77 are SNP sites and primer pairs within 2M downstream of the FANCA gene, and sequence numbers 77-105 are SNP sites and primer pairs within 2M upstream of the FANCA gene.

Table 1

Example 2

A couple was suspected of having a child with Fanconi anemia. The results of genetic testing indicated that the child's FANCA gene (NM_000135) had compound heterozygous mutations of c.3948delG (maternal) and c.2101A>G (p.Lys701Glu) (paternal). The couple applied for preimplantation genetic testing (PGT) for the above pathogenic mutations to give birth to healthy offspring. Due to insufficient genetic material of the child, blood samples from both parents of the couple were collected for genetic testing. Blood samples from both parents of the couple were collected for genetic testing, and the results indicated that the mutation carried by the female was inherited from the mother, and the mutation carried by the male was inherited from the father. The risk chromosome haplotypes of the male and female were clarified through family information.

The couple obtained 3 embryos (LYX-6, LYX-7 and LYX-8) through PGT. Trophoblast cell biopsy was performed when the embryos developed to the blastocyst stage. After whole genome amplification, the biopsied cells were further tested for known mutations and haplotypes of the FANCA gene. Of the 3 embryos, 2 were normal and 1 was a patient. The couple transplanted a normal embryo and underwent prenatal ultrasound diagnosis and genetic diagnosis at 16 weeks of pregnancy. B-ultrasound showed that the fetus developed normally, and genetic testing showed that the fetal FANCA gene did not have c.3948delG and c.2101A>G mutations, which was consistent with the PGT results. The specific testing steps are as follows:

1 Multiplex PCR capture: Mix the 105 primer pairs in Table 1 into two PCR reaction tubes, perform 105 multiplex reactions on the biopsy cell sample DNA in the two PCR reaction tubes, perform multiplex PCR amplification on the FANCA gene and upstream and downstream SNPs of the biopsy cell sample, and obtain the biopsy cell sample whole genome amplification product. The DNA content of each sample is greater than 500ng.

2. Build the library and sequence according to the Illumina standard library construction process.

The whole genome amplification products of the biopsy cell samples to be tested were constructed according to the Illumina standard library construction process and sequenced using Miseq.

When the DNA molecules to be tested come from multiple test samples, each sample can be added with a different label sequence (barcode) to distinguish the samples during the sequencing process, thereby achieving simultaneous sequencing of multiple samples.

3 Data analysis: trimmomatic software was used to remove the adapter sequence from the raw data generated by the Illumina sequencer, and the BWA software was used to align it to the human hg19 reference genome. BWA (Burrow-Wheeler-Aligner) was used to align the read segment with the reference genome sequence to obtain the position of the read segment on the reference genome. Finally, the haplotype SNP coverage and genotype of the sample to be tested were analyzed. The average sequencing depth reached 100x or more, which was considered qualified.

The results of the haplotype detection of the samples to be tested are shown in Table 2. The quality control results of the sequencing are shown in Table 3. The sequencing depth is above 100×, the 30× coverage is above 70%, and the 100× coverage is above 50%, and the quality control is qualified. Among them, F0 represents the male risk chromosome, F1 represents the male normal chromosome; M0 represents the female risk chromosome, and M1 represents the female normal chromosome.

Table 2

Name Haplotype Pathogenic gene testing (PGT-M) Female M0/M1 carry Male F0/F1 carry The man's mother F1/ normal Bride's mother M0/ carry LYX-6 M0/F0 Pathogenicity LYX-7 M1/F1 normal LYX-8 M1/F1 normal

Table 3

Table 4 shows the detection results of SNP mutations. Since the design scheme covers the entire coding region of the FANCA gene, gene mutations occurring in the coding region can be effectively detected, and the sequencing depth is greater than 100×.

Table 4

Note: *Heterozygosity: Hom means that the mutation site is a homozygous mutation, Het means that the mutation site is a heterozygous mutation, Hemi means that the mutation site is a hemizygous mutation, and Normal means that no mutation is detected at this site.

The SNP haplotypes are shown in Table 5, where “?” represents undetected, and the sites framed by black bold boxes are dropout sites. F0 represents the male risk chromosome, and F1 represents the male normal chromosome; M0 represents the female risk chromosome, and M1 represents the female normal chromosome.

Table 5

The samples of offspring embryos were verified by first-generation sequencing. The verification results are shown in Table 6, which are consistent with the PGT results.

Table 6

Name Generation Verification (c.3948delG) First Generation Verification (c.2101A>G) LYX-6 Het Het LYX-7 Normal Normal LYX-8 Normal Normal

Note: *Heterozygosity: Hom means that the mutation site is a homozygous mutation, Het means that the mutation site is a heterozygous mutation, Hemi means that the mutation site is a hemizygous mutation, and Normal means that no mutation is detected at this site.

Example 3

The method of Example 2 was used to test 16 human samples, and 16 FANCA carriers were detected. The mutation sites covered 43 exons of the FANCA pathogenic gene. The passing standard was set as no less than 2 effective SNPs on both sides of the gene, and the passing rate of the 16 subjects was 100%. This shows that the detection method of the FANCA gene mutation of the present application has high versatility and accuracy.

Since the number of single nucleotide polymorphism sites (SNPs) of the FANCA gene is large and widely distributed, the frequency of occurrence in the population exceeds 1%, and the total number reaches 3 million, with an average of 1/3Kb. The present application provides a pre-implantation genetic detection method, which can analyze 210 SNPs, detect a large number of SNPs, and has high versatility; 16 FANCA carriers were detected using the method of the present application, and the standard was set as no less than 2 effective SNPs on both sides of the mutation site, and the pass rate of the 16 subjects was 100%, with high versatility; the pre-experiment at the cell level was omitted, and the detection method of the present application can be easily automated on a high-throughput sequencing platform. Based on high-throughput sequencing technology, different label sequences are added to each sample, and a large number of samples can be analyzed at one time, reducing the cost of FANCA haplotype analysis; the detection method of the present application can be used to detect chromosome aneuploidy, chromosome structural abnormalities, and single-gene diseases, with a high sequencing depth, and an average sequencing depth of no less than 100X, which makes up for the defect that chip detection is easily affected by probes.

The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims (10)

1. A primer combination for detecting FANCA gene mutation, characterized in that the primer combination is selected from primer pair 1 to primer pair 105 with nucleotide sequences shown in SEQ ID No. 1 to SEQ ID No. 210. Many pairs. 2. The primer combination according to claim 1, characterized in that the primer combination includes primer pair 1 to primer pair 105 whose nucleotide sequences are shown in SEQ ID No. 1 to SEQ ID No. 210. 3. A kit for detecting FANCA gene mutation, comprising the primer combination according to claim 1 or 2. 4. The kit according to claim 3, characterized in that the kit further includes PCR reaction reagents. 5. The kit according to claim 4, wherein the PCR reaction reagents include one or more of PCR buffer, DNA polymerase, Mg ²⁺ and dNTPs. 6. A method for detecting FANCA gene mutations, characterized by comprising the following steps: Obtain the genomic DNA of the sample; Use the primer combination according to claim 1 or 2 or the kit according to any one of claims 3 to 5 to perform PCR amplification of the FANCA gene coding region of the genomic DNA of the sample and its upstream and downstream SNP sites; The amplified product was analyzed, and the FANCA mutation type was determined based on the analysis results. 7. The detection method according to claim 6, characterized in that the FANCA gene coding region and its upstream and downstream SNP sites include: chr16:86817040, chr16:86867197, chr16:86932609, chr16:86970454, chr16:86970479 , chr16:86970503, chr16:87019166, chr16:87060416, chr16:87060483, chr16:87111668, chr16:87232383, chr16:87305229, chr16:87343729, chr16:874 02133、chr16:87446368、chr16:87696272、chr16:87714622、chr16 :87720579、chr16:87722157、chr16:87727927、chr16:87730041、chr16:87738853、chr16:87742056、chr16:87748198、chr16:87748298、chr16:87748315、 chr16:87755154、chr16:87755155、chr16:87756047、chr16:87980060 , chr16:87980067, chr16:88038557, chr16:88080922, chr16:88081884, chr16:88081904, chr16:88081951, chr16:88085428, chr16:88089478, chr16:881 00321、chr16:88102208、chr16:88106046、chr16:88116559、chr16 :88164860、chr16:88164924、chr16:88165210、chr16:88165235、chr16:88453572、chr16:88460235、chr16:88469873、chr16:88504850、chr16:88520452、 chr16:88535620、chr16:88627084、chr16:88635395、chr16:88664758 , chr16:88668051, chr16:88680855, chr16:88791441, chr16:88822782, chr16:88880844, chr16:88902183, chr16:88902277, chr16:88909028, chr16:889 21022, chr16:89042334, chr16:89161684, chr16:89216941, chr16 :89304949、chr16:89437560、chr16:89568875、chr16:89590758、chr16:89600177、chr16:89649245、chr16:89675319、chr16:89697891、chr16:89800058、 chr16:89818732、chr16:89909429、chr16:89933420、chr16:89950299 , chr16:89954138, chr16:89957635, chr16:89958538, chr16:89961597, chr16:89967217, chr16:89972532, chr16:89975638, chr16:89985222, chr16:899 86154, chr16:89994413, chr16:90008296, chr16:90008896, chr16 :90013420、chr16:90020346、chr16:90024206、chr16:90024899、chr16:90031427、chr16:90044028、chr16:90046121、chr16:90060281、chr16:90074001、 chr16:90076227, chr16:90098466, chr16:90098616 and chr16:90107377 . 8. The detection method according to claim 6, wherein the method of analyzing the PCR amplification product includes: performing high-throughput sequencing on the PCR amplification product. 9. The detection method according to claim 6, characterized in that the sample is selected from one or more of peripheral blood, semen, oral mucosal cells and embryonic cells. 10. Use of the primer combination according to claim 1 or 2 or the kit according to any one of claims 3 to 5 for preparing a product for detecting FANCA gene mutations.