CN115058507A

CN115058507A - Primer group and kit for detecting folic acid metabolism related SNP (Single nucleotide polymorphism) marker

Info

Publication number: CN115058507A
Application number: CN202210237489.9A
Authority: CN
Inventors: 毛姗姗; 周栋明; 姚妹
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-03-11
Filing date: 2022-03-11
Publication date: 2022-09-16

Abstract

The invention relates to the technical field of gene detection, in particular to a primer group and a kit for detecting folic acid metabolism related SNP markers, wherein the primer group comprises a first amplification primer group or a first extension primer group, the first amplification primer group comprises primers with nucleotide sequences shown as SEQ ID NO. 3-8, and the first extension primer group comprises primers with nucleotide sequences shown as SEQ ID NO. 10-12. The invention also provides a primer group for jointly detecting the folic acid metabolism related SNP markers and the spinal muscular atrophy related gene copy number variation, wherein the primer group comprises the first amplification primer group, the second amplification primer group, the first extension primer group and the second extension primer group, the second amplification primer group comprises primers with nucleotide sequences shown as SEQ ID NO. 1-2, and the second extension primer group comprises primers with nucleotide sequences shown as SEQ ID NO. 9.

Description

Primer group and kit for detecting folic acid metabolism related SNP (Single nucleotide polymorphism) marker

Technical Field

The invention relates to the technical field of gene detection, in particular to a primer group and a kit for detecting folic acid metabolism related SNP markers.

Background

SMA (spinal muscular atrophy) is an autosomal recessive hereditary progressive motor neuron disease, characterized primarily by progressive degeneration of the anterior horn cells of the spinal cord and the motor nuclei of the brainstem. Clinically, it is mainly manifested as progressive, symmetrical muscle weakness and atrophy, with the proximal extremity heavier than the distal one and the lower extremity heavier than the upper one. The patient accounts for about 1/10000 in the newborn, and the carrier of the disease-causing gene accounts for about 1/50. According to the age of onset and clinical manifestations, the disease can be classified into 4 types, namely spinal muscular atrophy type I (SMN 1, also called Werdnig-Hoffmann disease) with onset less than 6 months, spinal muscular atrophy type II (SMN 2) with onset 6-18 months, spinal muscular atrophy type III (SMA 3, also called Kugelher-Welander disease) with onset in childhood or adolescent, and spinal muscular atrophy type IV (SMA 4) with onset in adults. The SMA pathogenic genes of the 4 subtypes are the same and are motor neuron survival gene 1 (SMN 1) [ OMIM600354 ]. The SMN1 gene is located on chromosome 5, has a total length of about 20kb and contains 9 exons. It is highly homologous to its immediate neighbors, SMN2 and SMN1, differing by only 5 nucleotides. SMN2 is a regulatory gene whose copy number is inversely proportional to the severity of the disease state of SMA.

Folic acid is an important nutrient required by organisms, is a water-soluble B vitamin and participates in a series of important biochemical reactions related to one-carbon unit transportation. Folic acid is an essential element for nucleic acid synthesis, is a necessary substance for cell growth and tissue repair, and is an indispensable nutrient in the process of embryonic development. Inadequate folic acid intake or folic acid metabolism disorder are important factors of newborn defects, and moreover, Hyperhomocysteinemia (HCY) caused by folic acid deficiency can also cause diseases such as maternal preterm delivery, gestational hypertension and the like; the insufficient folic acid intake or folic acid metabolic disorder can cause the male reproductive capacity to be reduced and other adverse effects on the father; for hypertensive patients, hypertension of the "H" type results, thereby increasing the risk of stroke. Therefore, the folic acid is reasonably supplemented, the HCY concentration in blood is reduced, and the folic acid is an effective measure for avoiding the occurrence of cerebral apoplexy. The A1298C site A → C mutation, C677T site C → T mutation and the 66A → G mutation of MTRR gene of MTHFR can affect the activity of enzyme related to folic acid metabolism, so that the utilization efficiency of folic acid is reduced, folic acid deficiency is easily caused, and the risk of diseases such as neural tube defect is increased.

In the prior art, MLPA, RT-PCR or NGS can be used for SMA and folic acid related gene mutation to a certain extent, but have certain defects. MLPA related detection can distinguish SMA patients, carriers and normal persons, detect SMN2 copy number of the patients, and detect 3 related mutation sites of folic acid metabolism at the same time, but the technical sample has high requirement, limited flux and more complex flow; signals are acquired by collecting fluorescence, so that signal deviation is easily caused; meanwhile, the point mutation can not be detected, and the cost is higher. The RT-PCR can distinguish SMA patients, carriers and normal persons, has low requirement on equipment, and has a fast detection flow and a fast detection speed compared with other two methods. However, RT-PCR signal interpretation utilizes amplification of fluorescent signals, which easily causes fluorescence interference. The NGS technique not only allows the targeted detection of SMA, but also allows the simultaneous coverage of other myopathies of similar phenotype. However, NGS can only be used as a primary screen, and the result of suspected positive needs to be verified by methods such as MLPA and the like. And the NGS has low accuracy and reliability in the aspect of SMN2 copy number analysis and high cost.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a primer set and a kit for detecting a folate metabolism-related SNP marker, which are used to solve the problems of the prior art.

In order to achieve the above objects and other related objects, the present invention provides a primer set for detecting a folate metabolism-related SNP marker, the primer set includes a first amplification primer set or a first extension primer set, the first amplification primer set includes primers having nucleotide sequences as shown in SEQ ID NOS: 3 to 8, and the first extension primer set includes primers having nucleotide sequences as shown in SEQ ID NOS: 10 to 12.

The invention also provides application of the primer group in preparation of SNP marker products related to folic acid metabolism.

The invention also provides a kit for detecting the folic acid metabolism related SNP marker, and the kit comprises the primer group.

The invention also provides a primer group for jointly detecting the folic acid metabolism related SNP markers and the spinal muscular atrophy related gene copy number variation, wherein the primer group comprises the first amplification primer group, the second amplification primer group, the first extension primer group and the second extension primer group, the second amplification primer group comprises primers with nucleotide sequences shown as SEQ ID NO. 1-2, and the second extension primer group comprises primers with nucleotide sequences shown as SEQ ID NO. 9.

The invention also provides application of the primer group for jointly detecting the folic acid metabolism related SNP marker and the spinal muscular atrophy related gene copy number variation in preparation of products for jointly detecting the folic acid metabolism related SNP marker and the spinal muscular atrophy related gene copy number variation.

The invention also provides a kit for jointly detecting the folic acid metabolism related SNP markers and the spinal muscular atrophy related gene copy number variation, and the kit comprises the primer group for jointly detecting the folic acid metabolism related SNP markers and the spinal muscular atrophy related gene copy number variation.

As described above, the primer set and the kit for detecting the SNP marker related to folate metabolism according to the present invention have the following beneficial effects: the Massa array technology is used for detecting SMA, the requirement on a sample is low, and whole blood DNA samples larger than 10 ng/mu l can be well detected and are suitable for common screening. Copy number detection and folate metabolism point mutation detection of the SMN1 gene are both completed in one reaction well, and interference among the reaction wells is eliminated. Through single base extension, the detection sites are distinguished by utilizing the difference of molecular weight among different bases, the type of the base at the detection sites can be directly and accurately detected, and the specificity is strong; fluorescent probes are not used, so that the fluorescent interference of similar sites is avoided, and the detection result is accurate. The experiment is completed in one reaction, a fluorescent probe is not used, and the experiment cost is low.

Drawings

FIG. 1-1 shows a MassaArray map of the detection sites on SMN1 exon7 in sample 800 (copy number 3).

FIG. 1-2 shows MassaArray maps of detection sites on SMN1 exon7 in sample 901 (copy number 2).

FIG. 2-1 shows MassaArray at the detection site rs1801131 of sample 908 (wild genotype).

FIG. 2-2 shows a MassaArray plot of rs1801131 detection site of sample 953 (heterozygous genotype).

FIGS. 2-3 show MassaArray at the detection site of rs1801131 in sample 904 (mutant genotype).

FIG. 3-1 shows MassaArray at the detection site of rs1801133 in sample 917 (wild type).

FIG. 3-2 shows MassaArray at the detection site of rs1801133 in sample 906 (heterozygous genotype).

FIGS. 3-3 show MassaArray's map of the detection site of rs1801133 in sample 952 (mutant genotype).

FIG. 4-1 shows MassaArray at rs1801394 detection site of sample 923 (wild type).

FIG. 4-2 shows MassaArray at rs1801394 detection site in sample 210 (heterozygous genotype).

FIGS. 4-3 show MassaArray at the detection site of rs1801394 in sample 917 (mutant genotype).

FIG. 5 shows the parameter settings on the mass spectrometer in the example.

Detailed Description

The invention provides a primer group for detecting a folic acid metabolism related SNP marker, which comprises a first amplification primer group or a first extension primer group, wherein the first amplification primer group comprises primers with nucleotide sequences shown as SEQ ID NO. 3-8, and the first extension primer group comprises primers with nucleotide sequences shown as SEQ ID NO. 10-12.

The SNP markers related to folate metabolism include MTHFR A1298C, MTHFR C677T and MTRR A66G.

MTHFR A1298C is also known as rs1801131 polymorphism, which causes the 429 th amino acid of the protein encoded by the MTHFR gene to be changed from Glu to Ala.

MTHFR A1298C includes 1298AA type (wild type), 1298AC type (heterozygous mutant) and 1298CC type (homozygous mutant).

MTHFR C677T is also known as rs1801133 polymorphism, which causes the amino acid number 222 of the protein encoded by the MTHFR gene to change from Ala to Val.

MTHFR C677T includes 677CC type (wild type), 677CT type (heterozygous mutant type) and 677TT type (homozygous mutant type).

MTRR A66G is also called rs1801394 polymorphism.

MTRR a66G includes 66AA type (wild type), 66AG type (heterozygous mutant), and 66GG type (homozygous mutant).

The specific base sequences of the amplification primer sets exemplified above may be replaced with 1 or more bases or 1 or more bases may be added to the 3 'end or the 5' end, as long as they can specifically recognize the respective specific recognition regions under the conditions for carrying out PCR (preferably, annealing and self-annealing do not occur between primers used in a single reaction vessel). The number of the plurality is, for example, 2 to 3. When 1 or more bases are added to the primer, it is preferable to add the base to the 5' end of the primer.

The identity of a nucleotide sequence obtained by substituting 1 or more nucleotides in a specific nucleotide sequence of the primer set exemplified above for other nucleotides is preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, and more preferably 95% or more, with the nucleotide sequence before substitution (i.e., the nucleotide sequence represented by the sequence number).

The length of each primer is not particularly limited as long as it can specifically recognize the corresponding specific recognition region and hybridization does not occur between the primers, and is preferably 15 bases or more and 40 bases or less. The lower limit of the length of the primer is more preferably 16 bases or more, still more preferably 17 bases or more, and still more preferably 18 bases or more. More preferably, the upper limit of the length of the primer is 39 bases or less, still more preferably 38 bases or less, and still more preferably 37 bases or less.

In one embodiment, the kit further comprises a PCR amplification reaction reagent, an alkaline phosphatase treatment reagent, and a single base extension reaction reagent.

In one embodiment, the PCR amplification reaction reagents include a DNA polymerase, a PCR buffer, a dNTP mix, and an aqueous medium.

The DNA polymerase is Taq DNA polymerase and/or high fidelity PCR enzyme, etc.

The buffer can generally provide the most suitable conditions for the enzymatic reaction in the PCR system. The buffer solution may be any buffer solution as long as it has the above-described effects.

The dNTP mixture is usually used as a raw material for DNA synthesis, and specifically may include dATP, dGTP, dTTP, dCTP, and the like.

The aqueous medium may be used to adjust the concentration of the components of the PCR system, and may generally act as a dilution solvent. In one embodiment, the aqueous medium is selected from ddH ₂ O。

Other various reagents required for nucleic acid amplification may also be included in the kit. For example, an amplification accelerator, magnesium ion may be included. Suitable amplification promoters may further improve non-specific amplification. For example, the amplification enhancer may be selected from a combination of one or more of betaine, SSB, DMSO, and the like. As another example, the concentration of the amplification enhancer may be 0.5-2.5M, 0.5-1M, 1-1.5M, 1.5-2M, or 2-2.5M. The magnesium ions can be generally used as a catalyst in a PCR system, can form dNTP-Mg with dNTP to interact with a nucleic acid skeleton, and can influence the activity of the Polymerase.

In one embodiment, the alkaline phosphatase treatment agent comprises SAP, an aqueous medium, a buffer. In one embodiment, the aqueous medium is selected from ddH ₂ O。

In one embodiment, the kit further comprises reagents for extracting genomic DNA from the sample. In one embodiment, the reagent for extracting genomic DNA from a sample may be an existing kit.

In one embodiment, the single base extension reaction reagent comprises an iPLEX enzyme, a buffer solution, an aqueous medium and a stop solution.

The variation sites of the copy number of the Spinal Muscular Atrophy (SMA) -related gene comprise SMN1E7: NM-001297715.1: c.840T or SMN 2E 7: NM-022875.3: c.840T. The normal copy number of the sites is 2, and the copy number after mutation is more than 3.

The kit also comprises a PCR amplification reaction reagent, an alkaline phosphatase treatment reagent and a single base extension reaction reagent. The nature of the PCR amplification reaction reagent, the alkaline phosphatase treatment reagent and the single base extension reaction reagent can be consistent with the corresponding reagents in the aforementioned kit for detecting the SNP marker related to folic acid metabolism.

The combined detection of the folic acid metabolism related SNP marker and the spinal muscular atrophy related gene copy number variation comprises the following steps:

s1, extracting DNA from a sample;

s2, performing multiplex PCR amplification reaction by using the amplification primers and the DNA in S1;

s3, carrying out dephosphorylation reaction on the amplification product obtained in the step S2;

s4, performing extension reaction on the product obtained in the step S3 by using the extension primer;

s5, purifying the product obtained in the step S4;

s6, measuring the extension primer of the purified product obtained in the step S5 by a Massa array technology to obtain the base type or gene copy number of the detection site;

s7, judging whether the patient has the risk of folic acid deficiency or not based on the base type detected in the step S6; based on the gene copy number detected in step S6, it is determined whether the patient has SMA.

The SMN1 gene is directly detected when the SMA is detected by the method, the copy number can be calculated through the peak height without detecting the changes of NAIP, H4F5 and GTF2H2 genes. The relative copy number of the gene does not need to be calculated according to the peak heights of two reference and target genes of the sample to be detected and the reference sample.

In one embodiment, the sample is from a human, preferably from a body fluid, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, lung lavage fluid, cerebrospinal fluid, stool, and tissue sample. More preferably fresh or frozen anticoagulated blood.

The basic principle of the MassARRAY technology system is a matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology, and the system has extremely high specificity and sensitivity. The system uses matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry to accurately detect DNA molecules. Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) is a novel biological Mass Spectrometry developed in recent years, the technology adopts a soft Ionization mode to generate stable molecular ions, the stable molecular ions fly to a detector under the action of an external electric field, and the Flight Time and the charge/Mass ratio (charge/molecular weight) of sample molecules form a certain proportional relation so as to obtain the accurate molecular weight of a substance to be detected, thereby being an effective method for determining the molecular weight of biological macromolecules. A DNA fragment has its own molecular weight, when a base is added or reduced in the sequence, even if one base is replaced by another base, its molecular weight is changed, MALDI-TOF-MS is the most sensitive technique for detecting this difference, its accuracy is up to 0.1% -0.01%, a mass spectrum peak is corresponding to DNA molecule with a certain molecular weight to realize qualitative identification, and its peak height is also proportional to the amount of this DNA molecule to realize quantitative analysis. Genetic variations are distinguished by analyzing their individual masses, eliminating the need for fluorescence or labeling.

Compared with the RT-PCR technology and the MLPA technology, the MassARRAY technology has the following advantages that:

1. relative to RT-PCR technology

1) The signal interpretation of the RT-PCR technology is to utilize the amplification of a fluorescent signal, eliminate the interference of similar sites by methods such as competitive probes and the like, and is difficult to effectively eliminate the interference, thereby causing data deviation and great interpretation difficulty.

The Massa array technology is used for detecting, and the difference of molecular weight among different bases is utilized to distinguish detection sites, so that the type of the base of the detected site can be directly and accurately detected. Fluorescent probes are not used, and fluorescent interference of similar sites is avoided.

2) RT-PCR technology can only detect one fragment in one reaction tube, and multiple detections require multiple reaction tubes. Easily cause the difference between reaction holes, and has low cost and limited detection flux.

The Massa array technology can complete the detection of all the sites in one reaction hole, thereby avoiding the difference between different reaction holes, reducing the cost and having large detection flux.

2. Compared with MLPA technology

1) MLPA technology has strict requirements on template quality, the amplified probe is a fluorescent probe, signal intensity is obtained by collecting fluorescence, the cost is high, signal deviation is caused, and adjacent fragments need to be considered when CNV is judged.

The Massa array technology has low requirements on sample quality, and the lower limit of genome DNA detection is 10 ng/mul. The difference of molecular weight among different bases is used for distinguishing detection sites, and the base type of the detected detection sites can be directly and accurately detected. Fluorescent probes are not used, so that the fluorescent interference of similar sites is avoided, and the cost is low.

2) In the MLPA technology, one reaction hole can only judge the copy number, and other sites such as mutation and the like cannot be judged, so that the flux is limited, and the process is complex.

The Massa array technology completes the detection of all the sites in one reaction hole, and has low cost and large detection flux.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Examples

1. MassaArray reaction mixed liquor preparation

1.1 PCR primer mixture preparation

PCR Primer MIX (containing 8 primers in one reaction well) is configured as follows:

1.2 preparation of PCR reaction mixture

The PCR reaction mixture system is shown in the following table, wherein 10 times PCR buffer and MgCl ₂ dNTP Mix, PCR Enzyme were purchased from Agena Bioscience.

1.3 preparation of SAP reaction mixture

The SAP reaction mixture system is shown in the following table, wherein SAP Buffer and SAP Enzyme are both available from Agena Bioscience:

ddH ₂ O	1.53μl
		SAP Buffer	0.17μl
SAP Enzyme	0.3μl
		total	2μl

1.4 preparation of extension primer mixture

Table below, iPLEX Primer MIX configuration table:

1.5 preparation of extension reaction mixture

The extension reaction mixture system is shown in the following table, in which iPLEX Buffer Plus, iPLEX Termination Mix, and iPLEX Pro Enzyme are all available from Agena Bioscience

ddH ₂ O	0.62μl
		iPLEX Buffer Plus	0.2μl
iPLEX Termination Mix	0.2μl
		iPLEX Primer MIX	0.94μl
iPLEX Pro Enzyme	0.04μl
		total	2μl

2. Genomic DNA acquisition

Fresh or frozen anticoagulated blood samples, wherein the samples are obtained from child hospitals affiliated to medical colleges of Zhejiang university, and genomic DNA is extracted by using a Ranunculus armomagnetic blood genomic DNA extraction kit according to the instruction.

gDNA was a 2 copy control, Human Genomic DNA purchased from Promega, cat # G1471. Normally, the SMN1 gene and SMN2 gene are 2 copies.

3. Multiple amplification step

3.1 PCR amplification step

And (3) uniformly mixing the PCR reaction mixed liquid prepared by 1.2, subpackaging the mixture into reaction wells corresponding to 384-well plates, subpackaging 4 mu l of each well, and then adding 1 mu l of the sample to be detected. Control gDNA and blank water were included at 2 copies for each assay. The membrane was adhered, centrifuged slightly, and then placed on a gene amplification apparatus to amplify the target DNA according to the PCR procedure shown in the following table.

3.2 SAP purification step

Mu.l of SAP reaction mix (1.3 configuration) was added to each well of 3.1 PCR-finished PCR amplification reaction, and the mixture was attached to a film, centrifuged slightly, placed on a gene amplifier, and purified according to the following SAP procedure:

37℃	40min
		85℃	5min
8℃	Hold

3.3 elongation reaction step

Mu.l of extension reaction mixture (1.5 configuration) was added to 3.2 SAP-finished SAP per well of SAP purified reaction, attached to a membrane, centrifuged slightly, placed on a gene amplifier, and extended according to the following extension reaction protocol:

4. desalting treatment and mass spectrometer spectrum drawing extension

After the reaction procedure, the reaction mixture was centrifuged instantaneously. Add 16. mu.L of sterile double distilled water, 6mg of clean Resin (Resin) to each well. Mix by inversion for 15min, centrifuge at 3200 Xg for 5 min. Samples were spotted and scored according to the parameters shown in FIG. 5.

5. Data analysis and interpretation of test results

5.1 interpretation of genotype results at SNP detection sites

Original files exported by instrument matching software Typers 4.0, xml files, path View → plate data disc; the results in the Call column are the results of the genotype at the SNP detection sites. The folate SNP typing results are given in the following table:

5.2 interpretation of SMN2 or SMN1 copy number test results

According to the result interpretation methods described in 5.1 and 5.2, the copy number variation of the SMN1 gene and the typing of 3 related SNPs in folate metabolism of each sample shown in FIGS. 1-1 to 4 can be determined. The sample 800SMN1 shown in FIG. 1-1 has a copy number of 3, which suggests that an MLPA double check should be made. The copy number of the sample 901SMN1 shown in fig. 1-2 was 2, i.e., it was determined that the SMN1 was not mutated. The SNP genotype of the site rs1801131 related to the folate metabolism of the sample 908 shown in FIG. 2-1 is TT, namely the wild type, the MTHFR enzyme activity is normal, and the risk of folate deficiency does not exist; in the sample 953 shown in fig. 2-2, the genotype of the SNP at the rs1801131 locus related to folic acid metabolism is GT, namely heterozygote, the activity of MTHFR enzyme is slightly reduced, and the risk of folic acid deficiency is possibly caused; 2-3, the SNP genotype of the rs1801131 locus related to the folate metabolism of the sample 904 is GG, namely mutant type, MTHFR enzyme activity is reduced, and the risk of folate deficiency is possibly existed; the same can be used to determine the results of FIGS. 3-1 through 4-3.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

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Claims

1. The primer group for detecting the folic acid metabolism related SNP markers is characterized by comprising a first amplification primer group or a first extension primer group, wherein the first amplification primer group comprises primers with nucleotide sequences shown as SEQ ID NO. 3-8, and the first extension primer group comprises primers with nucleotide sequences shown as SEQ ID NO. 10-12.

2. The primer set according to claim 1, wherein the folate metabolism-related SNP markers comprise MTHFRA1298C, MTHFR C677T and MTRR A66G.

3. Use of the primer set according to claim 1 or 2 in the preparation of a product for detecting a folate metabolism-related SNP marker.

4. A kit for detecting a SNP marker associated with folate metabolism, comprising the primer set of claim 1 or 2.

5. The kit of claim 4, wherein the kit further comprises a PCR amplification reaction reagent, an alkaline phosphatase treatment reagent, or a single base extension reaction reagent.

6. The kit of claim 5, further comprising any one or more of the following features:

1) the PCR amplification reaction reagent comprises DNA polymerase, PCR buffer solution, dNTP mixture and aqueous medium;

2) the alkaline phosphatase treatment agent comprises SAP, an aqueous medium and a buffer;

3) the single-base extension reaction reagent comprises iPLEX enzyme, buffer solution, aqueous medium and stop solution;

4) the kit also includes reagents for extracting genomic DNA from a sample.

7. A primer set for jointly detecting a folate metabolism-associated SNP marker and a spinal muscular atrophy-associated gene copy number variation, wherein the primer set comprises the primer set of claim 1 or 2, and a second amplification primer set and a second extension primer set; the second amplification primer group comprises primers with nucleotide sequences shown as SEQ ID NO. 1-2, and the second extension primer group comprises primers with nucleotide sequences shown as SEQ ID NO. 9.

8. The primer set of claim 7, wherein the spinal muscular atrophy-related gene copy number variation sites comprise SMN1E7: NM-001297715.1: c.840T or SMN 2E 7: NM-022875.3: c.840T.

9. Use of the primer set according to claim 7 or 8 in preparation of products for combined detection of folic acid metabolism-related SNP markers and spinal muscular atrophy-related gene copy number variations.

10. Kit for combined detection of folate metabolism-related SNP markers and spinal muscular atrophy-related gene copy number variations, comprising the primer set of claim 7 or 8.

11. The kit of claim 10, further comprising PCR amplification reagents, alkaline phosphatase treatment reagents, or single-base extension reagents.