A kit for integrating SMN1 and SMN2 copy number, minor variants and family linkage analysis and its application

Abstract

The present invention discloses a kit for integrating the copy numbers of SMN1 and SMN2, micro-variations and family linkage analysis, and its application. In the PCR reaction of the present invention, the exons of the SMN gene, SMN2 modifiers, SMN1 micro-pathogenic variations and molecular genetic markers are simultaneously amplified. The PCR products are analyzed by capillary electrophoresis and calculated with SMA normal controls in which the SMN gene is 2 copies, and the relative quantification detection of the copy numbers of exons 7 and 8 of the SMN1 and SMN2 genes is carried out, and the SMN genes with 0, 1, 2, 3, and ≥4 copies are calculated, differentiating the normal, heterozygous and homozygous deletion mutations of the SMN gene exon copy numbers, which can indicate the increase of SMN2 transcript, the presence of SMN1 micro-pathogenic variations, and provide the information required for family analysis to clarify the genetic pathway of the SMN1 gene. Starting from a DNA sample, the results can be obtained within 2.5 hours, and only one PCR experiment is required to achieve the auxiliary diagnosis covering all genetic variation modes of SMA. The detection technical process is simple and easy to achieve standardization.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a kit integrating SMN1 and SMN2 copy number, micro-variation and family linkage analysis and its application. Background Art

[0002] Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease caused by the degeneration of spinal cord anterior horn and medulla oblongata motor neurons, resulting in progressive symmetric muscle weakness and atrophy of proximal limbs and trunk. It is caused by the deletion or mutation of the survival motor neuron 1 (SMN1) gene located on chromosome 5q11.2-q13.3, which leads to a decrease in the expression of survival motor neuron protein (SMN), ranking first among lethal genetic diseases in children under 2 years old. The incidence of SMA in surviving newborns is about 1 / 6000 - 1 / 10000, but the carrier rate in the Chinese population is as high as 1 / 42. In 2018, it was included in the "First Batch of Rare Disease Catalogs" in China.

[0003] SMA is a single-gene genetic disease, but its genetic variation mode is very diverse. Approximately 95% of patients have homozygous deletion of exon 7 and / or exon 8 of SMN1, that is, the SMN1 copy number is 0. Another 5% are compound heterozygous mutant single-copy patients caused by point mutations and compound allele deletions of SMN1, all of which can affect the transcription of full-length mRNA and lead to a decrease in the level of SMN protein in the body, resulting in disease. Currently, among the micro-pathogenic variations reported in China, the detection frequency is ≥2, and there are a total of 7 micro-variations confirmed to be pathogenic by the pathogenicity assessment of the American College of Medical Genetics and Genomics (ACMG), including c.22dupA, c.683T>A, c.689C>T, c.863G>T, c.400G>A, c.463_464delAA, c.835-5T>G.

[0004] Furthermore, for SMA carriers, in addition to the common genotype of SMN1 "1+0", there are still nearly 8% of silent carriers, including SMN1 "2+0" and SMN1 "1+1" d " type. The SMN1 copy number of such carriers is ≥2, which is exactly the same as that of normal people, bringing many complexities to genetic counseling. Based on the 25% autosomal recessive genetic recurrence probability and the high carrier rate in the Chinese population, if accurate diagnosis is not given in time, at least 5000 new children will be inevitably added every year across the country, bringing a heavy burden to society.

[0005] SMN2 is a highly homologous gene to SMN1, with only 5 base differences between them. However, due to the c.840C>T mutation in exon 7 of SMN2, the structure of the splicing enhancer can be disrupted, resulting in exon 7 skipping of SMN2. Subsequently, an unstable truncated protein (SMNΔ7) that is easily degraded is encoded, and finally only a small amount of functional SMN protein is expressed to maintain physiological functions. As the most important biomarker in the disease process of SMA, the copy number of SMN2 is negatively correlated with the disease severity. The higher the copy number, the relatively milder the disease phenotype. At the same time, some studies have found that some SMA patients carrying 2 copies of SMN2 are not severely type 1 but milder type 2 or type 3. Further analysis found that these patients have rare polymorphic variations (such as c.859G>C, c.835-44A>G, etc.) on SMN2. Functional experiments have confirmed that these variations can lead to an increase in the full-length transcript containing exon 7 and an increase in the SMN protein level, thereby alleviating the disease progression and prognosis. Therefore, both SMN1 and SMN2 are elements in the genetic testing of SMA. Achieving the accurate detection of SMN1 and SMN2 at one time is very important for the diagnosis and prognosis of patients, and is even more significant for genetic counseling of carriers and the entire family.

[0006] Internationally, the common genetic testing method for SMA - Multiplex ligation-dependent probe amplification (MLPA) has certain limitations in the clinical applications of genetic diagnosis and carrier screening. Although this method can detect the copy number changes of up to 50 nucleotide sequences, identify mutations such as exon deletions and duplications in the same reaction tube, clearly distinguish patients, carriers, and normal individuals, and simultaneously detect the copy number of SMN2, it has high detection costs, a cumbersome detection method, takes at least 7 working days, and the experimental results have poor repeatability. It cannot detect about 5% of SMA patients with compound heterozygous mutations, easily causing missed diagnoses of SMA and false negatives in genetic counseling. Therefore, to achieve rapid and economical accurate diagnosis, it is urgent to develop new detection methods to meet the multi-level clinical needs.

[0007] Due to the existence of patients with compound heterozygous mutations and recessive carriers, the existing SMA detection technologies at home and abroad cannot simultaneously meet the rapid and accurate diagnosis and screening needs of all patients and carriers. Disease missed diagnoses are extremely likely to occur, which can even lead to false negatives in genetic counseling and increase birth defects. In view of this, a modified method is needed to simultaneously detect the copy numbers of SMN1 and SMN2, the minor variations of SMN1, the modifiers of SMN2, and the molecular genetic marker (short tandem repeat, STR) for family linkage analysis of the SMN gene in an accurate, specific, and faster way to determine whether there is 2+0 or 1+1 dRecessive carriers, that is, a multiplex mutation amplification system fluorescence PCR technology covering all genetic variation modes of SMA, is used for pedigree linkage analysis to clarify the genetic pathway of the SMN1 gene, and truly achieve rapid and accurate diagnosis of SMA patients and carriers of all gene types. Summary of the Invention

[0008] The object of the present invention is to provide a kit for integrating the copy numbers of SMN1 and SMN2, detecting minor variations and performing pedigree linkage analysis, and its application, in view of the above problems.

[0009] To achieve the aforementioned invention object, the present invention adopts the following technical solutions:

[0010] A kit for integrating the copy numbers of SMN1 and SMN2, detecting minor variations and performing pedigree linkage analysis, the kit comprising:

[0011] 1) Primers for detecting the copy numbers of SMN1 and SMN2 genes, and the copy number detection sites of the SMN1 and SMN2 genes include the following: SMN1 exon 7, SMN1 exon 8, SMN2 exon 7, SMN2 exon 8;

[0012] 2) Primers for detecting minor variations of SMN1, and the minor variations of SMN1 include the following: c.22dupA, c.683T>A, c.689C>T, c.863G>T, c.400G>A, c.463_464delAA, c.835-5T>G;

[0013] 3) Primers for detecting SMN2 modifiers, and the SMN2 modifiers include the following: c.859G>C and c.835-44A>G;

[0014] 4) Primers for detecting short tandem repeat sequence sites on chromosome 5 near the SMN1 and SMN2 genes, and the short tandem repeat sequence sites include the following: D5S125, D5S629, D5S1414, D5S1408, D5S610, D5S112;

[0015] 5) Primers for detecting internal control genes, and the internal control genes include the following: CFTR, RPP30;

[0016] 6) Multiplex polymerase chain reaction (PCR) reagents.

[0017] Further, the kit sets corresponding wild-type detection primers for SMN1 micro-variations and SMN2 modifiers. The wild-types corresponding to the SMN1 micro-variations and SMN2 modifiers include the following: c.22A, c.683T, c.689C, c.863G, c.400G, c.463A, c.835-5T, c.859G, and c.835-44A.

[0018] Further, the primer sequences in the kit are specifically as shown in SEQ ID NO.1 to 49, and the specific sequences are shown in the following table.

[0019]

[0020]

[0021]

[0022] Further, the primers in the kit contain at least one kind of fluorescent group, and the fluorescent group is selected from any one of FAM, VIC, HEX, JOE, TMR, NED, PET, and ROX.

[0023] A method for detecting SMA genetic variations for non-disease diagnosis and treatment purposes, which uses the above-mentioned kit that integrates SMN1 and SMN2 copy numbers, micro-variations, and family linkage analysis.

[0024] Further, the above-mentioned method for detecting SMA genetic variations for non-disease diagnosis and treatment purposes includes the following steps:

[0025] (1) Mix the DNA sample to be tested and the primers shown in SEQ ID NO.1 - 49 in the PCR reagent;

[0026] (2) Subject the mixture obtained in step (1) to polymerase chain reaction, and perform fluorescence-labeled capillary electrophoresis analysis on the target fragment after the polymerase chain reaction;

[0027] (3) Use GeneMapper software to separate the amplification products with different fluorescence and different fragment lengths produced in step (2) through high-resolution technology, and generate detection peak area information;

[0028] (4) Determine whether the DNA sample to be tested has SMA genetic variations by judging the area of each detection peak.

[0029] Further, in the above method for detecting SMA genetic variations for non-disease diagnosis and treatment purposes, the SMA genetic variations include whether the copy numbers of exon 7 of SMN1, exon 7 of SMN2, exon 8 of SMN1, and exon 8 of SMN2 in the DNA sample to be tested are mutated, whether the DNA sample to be tested has SMN1 minor variations and SMN2 modifier genes, and whether the DNA sample to be tested is an SMN(2+0) genotype SMA recessive carrier.

[0030] Application of the above kit integrating SMN1 and SMN2 copy numbers, minor variations and family linkage analysis in SMA gene detection.

[0031] The beneficial effects of the present invention are as follows:

[0032] (1) Combine multi-stage PCR amplification reactions in the same tube to solve the problems of multiplex PCR amplification quantification and qualification at one time. Using various different types of primers in a single tube reaction solution, perform PCR amplification reactions, which takes 1.5 hours in total. At the same time, amplify the 7th exon and 8th exon regions of SMN1 and SMN2 genes, SMN2 modifier factors, common SMA minor pathogenic variations in China (detection frequency ≥ 2), and molecular genetic markers (STR) of SMN1 gene. Combine the original multi-stage PCR amplification reactions in the same tube to solve the multiplex PCR problem at one time, which can greatly shorten the detection time.

[0033] (2) Ensure the correctness of the detection results through the review of the total copy number. In addition to the 7th exon, simultaneously detect the 8th exon of SMN1 and SMN2 genes. Under normal circumstances, the total copy number of SMN1+SMN2 in the 7th exon and 8th exon of SMN1 and SMN2 should be the same. If the total copy number is found to be inconsistent, it indicates that there is a problem with PCR amplification, which can avoid false negative or false positive results leading to misjudgment.

[0034] (3) Simultaneously detect the pathogenic gene SMN1 of SMA and the clinical phenotype prediction gene SMN2, which can distinguish SMA patients, carriers, and normal people. For patients with 0 copy number of SMN1 gene, combined with the SMN2 gene copy number results and supplemented with the detection of SMN2 gene modifier factors (c.859G>C and c.835-44A>G), the severity of the disease can be evaluated and can be used for the prognosis assessment of clinical drug treatment.

[0035] (4) Simultaneously include the detection of common SMA minor pathogenic variations in China, which can supplement the deficiencies of copy number detection, fill the gap of 5% compound heterozygous mutation SMA patients, and avoid false negative results.

[0036] (5)Individuals with the 2+0 genotype of SMN1 are a special type of recessive carriers of SMA. Approximately 8% of carriers belong to this genotype, posing a great challenge to carrier screening and genetic counseling for families. Detection of the molecular genetic marker (STR) of the SMN1 gene can be used for family linkage analysis and is of great significance for SMA prevention within the entire family and confirmation of the SMN1(2+0) genotype.

[0037] (6)The single-tube PCR reaction of the present invention, combined with one-well capillary electrophoresis analysis, can produce results within 2.5 hours, quantitatively determining the copy numbers of SMN1 and SMN2 genes at one time, qualitatively detecting minor variations of the SMN1 gene, SMN2 modifiers, and the molecular genetic marker of the SMN gene. Among them, the detection results of the SMN1 copy number and pathogenic variations are used for diagnosis or differential diagnosis, and the detection results of the SMN2 copy number and modifiers are used as reference indicators for treatment, clinical management, and prognosis evaluation after patient diagnosis. The detection coverage rate of patients reaches 100%, which can accurately distinguish normal people, carriers, and patients with SMA, provide SMA phenotype prediction, simultaneously meet the clinical needs of multiple detections for the entire family, and is expected to further carry out neonatal screening. Therefore, it has important social significance for realizing the tertiary prevention of SMA, reducing birth defects, improving the quality of the population in China, and significantly reducing social medical costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The detection result of an SMA carrier sample, with SMN1:SMN2 being 1:1.

[0039] Figure 2 The detection result of a normal SMA sample, with SMN1:SMN2 being 2:2.

[0040] Figure 3 The detection result of a normal SMA sample, with SMN1:SMN2 being 3:3.

[0041] Figure 4 The detection result of a normal SMA sample, with SMN1:SMN2 being 4:2.

[0042] Figure 5 The detection result of an SMA pathogenic minor variation c.22dupA sample, with SMN1:SMN2 being 3:1. DETAILED DESCRIPTION OF THE INVENTION

[0043] Example 1 Primer Design

[0044] According to the NCBI database, specific primers were designed for the 7th and 8th exons of the SMN1 and SMN2 genes, 2 SMN2 modifiers, 7 SMN1 micro-pathogenic variants, 6 molecular genetic marker sites of the SMN gene, and the CFTR and RPP30 genes, as shown in SEQ ID NO.1 - 49. Among them, the 7th and 8th exons of SMN were used for SMN copy number determination, the SMN2 modifier indicated an increase in the SMN transcript in SMA patients, the SMN1 micro-pathogenic variants indicated that the sample was a possible carrier or patient, and two reference genes (CFTR and RPP30) were used for monitoring the PCR efficiency.

[0045] Preparation of SMA reaction solution and SMA primer mixture

[0046] Table 2 Composition table of SMA reaction solution

[0047] Component Final concentration per reaction 10X Taq Buffer (Thermo) 1.5X <![CDATA[25mM MgCl2]]> 0.6 mM dATP / dTTP / dGTP / dCTP 0.5 mM each Betaine 2.0M

[0048] Table 3 Composition table of SMA primer mixture

[0049]

[0050]

[0051] Example 2 PCR amplification and result analysis

[0052] 1. Sample treatment: Human genomic DNA was extracted using a nucleic acid extraction kit (Qiagen, QlAmp DNA Blood Mini Kit, product number 51104) for subsequent PCR reactions. The DNA concentration was 7.5 ng / μL - 60 ng / μL, and the ratio of OD 260 nm / OD 280 nm was between 1.6 - 2.0.

[0053] 2. Preparation of amplification reagents:

[0054] (1) Take out the SMA reaction solution and SMA primer mixture from the kit, thaw at room temperature, mix well by inverting up and down, and briefly centrifuge with a microcentrifuge to sediment all the liquid at the bottom of the tube.

[0055] (2) Preparation of amplification reagents: Prepare the amplification reagents according to Table 4 below

[0056] Table 4 Preparation table of amplification reagents

[0057] Amplification reagent Volume per reaction SMA reaction solution 15 μL SMA primer mixture 33 μL DNA hot start polymerase 0.1 μL Total volume 18.1 μL

[0058] (3) Add 18 μL of the prepared amplification reagent to each PCR reaction tube and transfer it to the sample treatment area for loading.

[0059] 3. Sample addition: Add 2 μL of the genomic DNA of the sample to be tested, normal control, and blank control respectively into the corresponding PCR reaction tubes containing 18 μL of amplification reagent. After covering the PCR reaction tube caps, centrifuge briefly.

[0060] 4. PCR amplification and capillary electrophoresis analysis:

[0061] (1) Place the PCR reaction tubes into the PCR instrument to set the reaction program, and set the reaction volume: 20 μL;

[0062]

[0063] (2) Fluorescently labeled capillary electrophoresis analysis of PCR amplification products: Use ABI3130, ABI3730, ABI3500Dx, or ABI SeqStudio Genetic Analyzer capillary gene analyzer for detection. Take 1 μL of the amplification product, 10 μL of 1% GeneScan 500LIZ Size Standard (prepared by adding 10 μL of GeneScan 500LIZ Size Standard to 990 μL of Hi-Di Formamide, which is 1 vol% GeneScan 500LIZ Size Standard), denature at 95 °C for 3 minutes, immediately place on ice for 2 minutes, and then load onto the machine for detection.

[0064] 5. Software analysis:

[0065] After the reaction program ends, use GeneMapper software to analyze the fragment sizes of the PCR amplification products. The required Analysis Method, Panel, and Size Standard files for analysis can be downloaded from the website. For specific information on data import, analysis parameter settings, and result analysis, please refer to the GeneMapper user manual.

[0066] 6. Calculation of detection peak ratio:

[0067] The detection peak areas of the SMA gene and two internal reference genes of the sample to be tested and the normal control are respectively divided by the total area of the internal reference genes, and then the sample to be tested is divided by the corresponding values of each site of the SMA female control, which is the detection peak ratio (R) of each site.

[0068] 7. Result interpretation:

[0069] (1) When the detection peak ratio (R) of the internal reference gene falls between 1.80 - 2.20, the copy number interpretation of the detection peak ratios of exons 7 and 8 of the SMN1 and SMN2 genes is as follows in the table.

[0070] Copy number determination Range of detection peak ratio (R) 0 R＜0.20 1 0.20≤R＜1.45 2 1.45≤R＜2.5 3 2.5≤R＜3.5 ≥4 R≥3.5

[0071] (2) When SMN1 has 0 or 1 copy with pathogenic compound heterozygous mutations, the SMN2 modifiers c.859G>C and c.835-44A>G loci have variant detection peak signals, which can indicate an increase in full-length SMN2 transcripts and, in combination with other clinical information, suggest the possibility of milder SMA symptoms.

[0072] (3) When the copy number of SMN1 is 1 copy and there are variant detection peak signals at the pathogenic variant sites of SMN1 (c.22dupA: 217bp, c.835-5T>G: 251bp, c.863G>T: 284bp, c.463_464delAA: 296bp, c.689C>T: 327bp, c.683T>A: 340bp, c.400G>A: 359bp), it can indicate the possible existence of the above pathogenic microvariants on SMN1, and comprehensive evaluation is required by combining other methodologies such as long-fragment amplification, third-generation sequencing, clinical information, and family history.

[0073] Example 3 Verification of Reagent Performance

[0074] 1. Evaluate the diagnostic accuracy of the reagent of the present invention

[0075] Detect 10 accuracy reference products (including 4 deletion reference products, 5 carrier reference products, and 1 microvariant reference product). As shown in the following table, detect high, medium, and low concentrations, with sample concentrations of 60 ng / μL, 25 ng / μL, and 15 ng / μL respectively. Each concentration is tested in triplicate, and three batches of reagents are detected. The exon copy number of the SMA gene meets the requirements.

[0076] Table 5 Gene copy numbers of exon 7 and exon 8 of the accuracy reference product

[0077]

[0078] 2. Coincidence rate of specific reference products

[0079] Detect 12 normal reference products. As shown in the following table, detect high, medium, and low concentrations, with sample concentrations of 60 ng / μL, 25 ng / μL, and 15 ng / μL respectively. Each concentration is tested in triplicate, and three batches of reagents are detected. The coincidence rate of the detection results for specificity is 100%.

[0080] Table 6 Gene copy numbers of exon 7 and exon 8 of the specific reference product

[0081]

[0082] 3. Repeatability

[0083] Three repeat reference samples were tested, with ten replicates for each concentration. Three batches of reagents were tested, and the copy number judgments were all correct, and the test results were all in line.

[0084] Table 7 Gene copy numbers of the 7th exon and 8th exon of the repeat reference sample

[0085]

[0086] 4. Detection limit

[0087] Thirteen detection limit reference samples were tested. As shown in the following table, the sample concentrations were diluted to 20 ng / μL and 15 ng / μL respectively, and 20 replicates were performed for each. Three batches of reagents were tested, and the number of replicates of the test results all met the requirements.

[0088] Table 8 Gene copy numbers of the 7th exon and 8th exon of the detection limit reference sample

[0089]

[0090] Example 4

[0091] 1. Collect 150 EDTA-anticoagulated whole blood samples, and use Probemix P021 developed by MRC-Holland, the Netherlands as the reference method to verify the consistency between the results. Probemix P021 as a reference method to verify the consistency between the results.

[0092] 2. Collect EDTA-anticoagulated whole blood samples, extract human genomic DNA using a nucleic acid extraction kit (Qiagen, QlAmp DNA Blood MiniKit, product number 51104), and detect the concentration and purity of DNA with a micro-spectrophotometer. The concentration of 150 samples is 15 ng / μL - 60 ng / μL, and the ratio of OD 260nm / OD 280nm is between 1.6 and 2.0.

[0093] 3. According to the steps of Example 2, perform DNA loading and react with a PCR instrument.

[0094] 4. According to the steps of Example 2, perform result analysis. The results are as shown in the following table, all meeting the requirements of the SMA exon copy number. Among them, 51 samples were tested for SMA patients, 22 samples were tested for SMA carriers, and 77 samples were tested for normal SMA individuals, with a total of 150 samples tested (Table 9).

[0095] Table 9 Test results of 150 samples

[0096]

[0097]

[0098]

[0099] *MLPA: Multiplex Ligation-dependent Probe Amplification, which serves as a positive control group in the present disclosure.

[0100] According to Table 9 and Figures 1 to 4 the results, the present invention can accurately determine the copy number of exons of the SMN gene in a human individual, and further identify spinal muscular atrophy patients, spinal muscular atrophy carriers, and normal types.

[0101] Example 5

[0102] 1. For SMN gene point mutation samples, 21 EDTA-anticoagulated whole blood samples were collected, and first-generation sequencing of the SMN gene was used as a reference method to verify the consistency between the results.

[0103] 2. EDTA-anticoagulated whole blood samples were collected, and human genomic DNA was extracted using a nucleic acid extraction kit (Qiagen, QlAmp DNA Blood MiniKit, product number 51104). The concentration and purity of the DNA were detected with a micro-spectrophotometer. The concentration of the 21 samples was 15 ng / μL - 60 ng / μL, and the ratio of OD 260nm / OD 280nm was between 1.6 and 2.0.

[0104] 3. According to the steps of Example 2, DNA was loaded, and the reaction was carried out with a PCR instrument.

[0105] According to the steps of Example 2, the results were analyzed. The results are shown in the following table, all of which meet the requirements for the copy number of SMA exons. Among them, 14 samples were detected for SMN gene c.22dupA, 2 samples were detected for c.683T>A, and 1 sample was detected for each of c.689C>T, c.863G>T, c.400G>A, c.463_464delAA, and c.835-5T>G. A total of 21 samples were detected (Table 10).

[0106] Table 10 Detection Results of 21 Samples

[0107]

[0108] It should be understood that the foregoing description of the embodiments is only given by way of examples, and those with ordinary knowledge in the technical field to which the present invention pertains can make various modifications. The above specification, examples and experimental results provide a complete description of the structure and use of the exemplary embodiments of the present invention. Although various specific embodiments of the present invention are disclosed in the above embodiments, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention pertains can make various changes and modifications without departing from the principles and spirit of the present invention. Therefore, all equivalent changes and modifications made in accordance with the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A kit for integrating SMN1 and SMN2 copy number, minor variants, and family linkage analysis, characterized in that: The kit comprises: 1) Primers for detecting the copy numbers of SMN1 and SMN2 genes, and the detection sites for the copy numbers of SMN1 and SMN2 genes include the following: SMN1 exon 7, SMN1 exon 8, SMN2 exon 7, SMN2 exon 8; 2) Primers for detecting the minor variations of SMN1, and the minor variations of SMN1 include the following: c.22dupA, c.683T>A, c.689C>T, c.863G>T, c.400G>A, c.463_464delAA, c.835-5T>G; 3) Primers for detecting the modifiers of SMN2, and the modifiers of SMN2 include the following: c.859G>C and c.835-44A>G; 4) Primers for detecting the short tandem repeat sequence sites on chromosome 5 near the SMN1 and SMN2 genes, and the short tandem repeat sequence sites include the following: D5S125, D5S629, D5S1414, D5S1408, D5S610, D5S112; 5) Primers for detecting the internal control genes, and the internal control genes include the following: CFTR, RPP30; 6) Multiple polymerase chain reaction reagents; The kit sets corresponding wild-type detection primers for the minor variations of SMN1 and the modifiers of SMN2, and the wild types corresponding to the minor variations of SMN1 and the modifiers of SMN2 include the following: c.22A, c.683T, c.689C, c.863G, c.400G, c.463A, c.835-5T, c.859G and c.835-44A; The primer sequences included in the kit are specifically shown as SEQ ID NO.1~49.

2. The kit for integrating the copy number of SMN1 and SMN2, micro-variation and pedigree linkage analysis according to claim 1, characterized in that: The primers contain at least one kind of fluorescent group, and the fluorescent group is selected from any one of FAM, VIC, HEX, JOE, TMR, NED, PET, ROX.

3. Use of the kit according to claim 1 in the preparation of a product for detecting SMA gene.

4. A method for detecting SMA genetic variations for non-diagnostic and non-therapeutic purposes, characterized in that: Performed using the kit according to claim 1.

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