Method for detecting human motor neuron survival gene SMN

Abstract

The invention discloses a method for detecting a human motor neuron survival gene SMN and a kit for detecting the human motor neuron survival gene SMN. The invention relates to a calculation method capable of better performing copy number variation of a human motor neuron survival gene SMN and application of the calculation method in accurate and quantitative detection of the high copy number of the human motor neuron survival gene SMN. By adopting the method disclosed by the invention, the copy number variation of SMN1 exon 7 and exon 8, SMN2 exon 7 and exon 8, SMN1 tiny variation, composite heterozygous mutation and silence carriers can be simultaneously detected in one tube.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting human motor neuron survival gene SMN. Background Art

[0002] Spinal muscular atrophy (SMA) is the most common autosomal recessive neuromuscular disease in children, with muscle weakness and atrophy caused by degeneration of α-motor neurons in the anterior horn of the spinal cord as the main clinical features. The incidence of SMA is about 1 / 10,000, and the carrier rate in Asian population is about 1 / 48~50.

[0003] The genes associated with the onset of SMA are located in the 5q13.2 region of human chromosome 5, namely the survival motor neuron (SMN) gene, which includes the pathogenic gene SMN1 and the modifier gene SMN2. These two genes are highly homologous and reversely repeated. This feature makes the SMN gene prone to recombination or deletion.

[0004] The deletion, transformation or mutation of the pathogenic gene SMN1 is the main factor in the onset of SMA. 95% of SMA patients are caused by homozygous deletion of the pathogenic gene SMN1, that is, [0+0] type, and another 5% are caused by compound heterozygous mutation of SMN1, that is, [0+1d] type. d The phenotype is composed of a homozygous deletion of one chromatid and a point mutation in the other chromatid that affects the expression of the SMN1 gene. Currently, there are two mutation sites unique to Chinese people found in large-sample studies in China: one is located on exon 1. mutation, resulting in a frameshift mutation; the other is located on exon 5 These two mutations were confirmed as highly pathogenic sites by the American College of Medical Genetics and Genomics (ACMG) pathogenicity assessment.

[0005] According to the copy number of SMN1, SMA patients, carriers and normal people can be distinguished: when the SMN1 copy number of the subject is 0, it is an SMA patient; when the SMN1 copy number of the subject is 1, it is an SMA carrier. If it also carries a minor mutation, the subject is a compound heterozygous mutation; when the SMN1 copy number of the subject is 2, each chromosome carries 1 copy, that is, the [1+1] type, which is a normal person, but for a special type of carrier (the frequency in the carrier population is about 4%), the two copies are located on the same chromosome, and the SMN1 gene on the other chromosome is lost, that is, the [2+0] type, which is called a silent carrier. There is a 50% probability that the chromosome without SMN1 will be passed on to the offspring, resulting in SMA patients. For the [2+0] type detection, the early strategy was to conduct family analysis by introducing several molecular genetic markers. This operation method is very cumbersome and not suitable for screening. In addition, the primer complexity of such a system is higher, and its amplification stability will be affected. There are literature reports (Luo et al. Genet Med 2014, 16:149–156) on polymorphic sites of the SMN1 gene Closely related to the [2+0] silent carrier type, that is, when the subject has 2 copies of SMN1 and carries When a point mutation occurs, the subject is a [2+0] type silent carrier, which provides a new idea for the rapid identification of the [2+0] type.

[0006] About 80-90% of the proteins produced by the modified gene SMN2 are truncated and unstable, and another 10-20% of the full-length proteins have normal functions, which can partially compensate for the gene function lost by SMN1 due to mutation. In the domestic and international management consensus, SMA is generally divided into types I-IV according to the patient's SMN2 copy number, age of onset, and motor ability. The SMN2 copy number increases gradually, and the age of onset and the severity of the disease decrease gradually. Therefore, the SMN2 copy number is often used as an important reference data in clinical trials of drugs that use the regulation of SMN protein as a treatment strategy, and in newborn screening, it is used as an important biological marker for the evaluation of patient treatment before symptoms appear.

[0007] It can be seen that in addition to the copy number of the SMN1 gene, the mutation of the key sites of the SMN1 gene is of great value for screening the normal population. At the same time, the precise quantification of the copy number of the SMN2 gene is also of great guiding significance for clinicians to formulate corresponding treatment plans for different patients. In clinical practice, a fast and convenient detection technology or product that can meet the above needs at the same time is needed.

[0008] At present, most of the SMA mutation detection technologies reported and marketed at home and abroad rely on real-time fluorescence quantitative PCR (real-time PCR). This technology has a short detection cycle and a high penetration rate of instruments, which is suitable for population screening. However, it does not include the detection of hotspot mutation sites, which will cause the missed detection of patients with compound heterozygous mutations or [2+0] silent carriers, causing irreparable harm to patients and their families. In addition, it is impossible to accurately quantify the copy number ≥ 2. The copy number detection of SMN1 and SMN2 requires separate multi-tube reactions, which is complicated and inconvenient to operate. Multiplex ligation-dependent probe amplification (MLPA) based on capillary electrophoresis platform is the gold standard for detecting SMN1 and SMN2 copy number variations recommended by consensus at home and abroad. It can not only achieve accurate quantification of copy number, but also complete the detection of point mutations. However, this method is not only expensive, cumbersome to operate (it needs to go through denaturation, hybridization, ligation, amplification, machine detection and other steps), and time-consuming (it takes 2 days to complete the entire process detection), but it cannot meet the needs of large-scale screening.

[0009] Fluorescence PCR-capillary electrophoresis is a technique that uses fluorescent dyes to label primers, amplifies PCR products in capillary lanes, and uses software to collect images and analyze the size of amplified fragments. The earliest patent that attempted to use this technology to simultaneously detect SMN copy number and mutations in each exon (patent document US7875432B2) compared to other detection technologies is that the method has the advantages of relatively simple operation and short detection cycle. The disadvantage is that although it can detect copy number and point mutations at the same time, it needs to be amplified in two systems, and can only obtain the copy number ratio relationship between SMN1 and SMN2, and cannot accurately quantify the copy number of SMN1 and SMN2. However, this attempt makes it possible to detect the SMN gene based on fluorescence PCR-capillary electrophoresis technology. Later, an improved method for detecting SMN based on this technology emerged (see Chinese patent document CN112048548B). The improved method can quantify the copy number and key point mutations of SMN1 and SMN2, but it still has shortcomings: the result of detection by one system is the sum of the copy numbers of exon 7 and exon 8 of SMN1 and SMN2 genes, while point mutations are detected by another system.

[0010] Therefore, there is no convenient, fast, and affordable product that can not only detect the copy number of SMN1 and SMN2 in one tube, but also detect key mutation sites to distinguish compound heterozygous mutations and reliably distinguish SMN1 [2+0] silent carriers from SMN1 [1+1] normal people, which can not only meet clinical diagnostic needs but also screen carriers. Summary of the invention

[0011] In order to solve the shortcomings of the current methods for detecting the copy number of SMA-related genes, compound heterozygous mutations and silent carriers, the present invention screened and compared a large number of alternative internal references, and finally selected one or two of the hemoglobin subunit beta Gene (HBB) gene and the ribonuclease P / MRP subunit p30 (RPP30) as internal references to calculate the results, and through the screening of a large number of primers and system optimization, it is achieved in one tube that SMN1 and SMN2 exon 7 and exon 8, two high-frequency mutation sites in the Chinese population and key mutation points for distinguishing [2+0] type silent carriers are amplified simultaneously. Such a system design can achieve high-throughput detection of multiple targets at a lower detection cost, which is more in line with clinical needs.

[0012] Therefore, the first object of the present invention is to provide a method for detecting the human motor neuron survival gene SMN. The second object of the present invention is to provide a kit for detecting the human motor neuron survival gene SMN. The third object of the present invention is to provide a calculation method that can better detect the copy number variation of the human motor neuron survival gene SMN. The fourth aspect of the present invention is the application of the calculation method in the accurate quantitative detection of the high copy number of the human motor neuron survival gene SMN.

[0013] To achieve the above object, the present invention adopts the following technical solutions: As a first aspect of the present invention, a method for detecting the survival motor neuron gene SMN in human is provided, which comprises a primer combination 1 capable of specifically amplifying SMN1 and SMN2 distinguishing exon 7 sites, a primer combination 2 capable of specifically amplifying SMN1 and SMN2 distinguishing exon 8 sites, a primer combination 3 capable of specifically amplifying a site containing a mutation site c.22dupA, a primer combination 4 capable of specifically amplifying a site containing a mutation site c.683T / A, and a primer combination 5 capable of specifically amplifying a site containing a mutation site The primer combination 5 is used for amplification to obtain an amplified product; and amplifying with an internal reference primer HBB and / or an internal reference primer RPP30 to obtain an internal reference HBB amplification product and / or an internal reference RPP30 amplification product; By calculating the ratio of the peak height of the amplified product of exon 7 or exon 8 of SMN1 or SMN2 to the peak height of the amplified product of internal reference HBB, or, By calculating the ratio of the peak height of the amplified product of exon 7 or exon 8 of SMN1 or SMN2 to the peak height of the amplified product of the internal reference RPP30, or, By calculating the ratio of the peak height of the amplified product of exon 7 or exon 8 of SMN1 or SMN2 to the sum of the peak height of the amplified product of the internal reference HBB and the peak height of the amplified product of the internal reference RPP30, or, By calculating the sum of the ratio of the peak height of the amplified product of exon 7 of SMN1 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 7 of SMN1 to the peak height of the amplified product of internal reference RPP30, and / or, By calculating the sum of the ratio of the peak height of the amplified product of exon 8 of SMN1 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 8 of SMN1 to the peak height of the amplified product of internal reference RPP30, and / or, By calculating the sum of the ratio of the peak height of the amplified product of exon 7 of SMN2 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 7 of SMN2 to the peak height of the amplified product of internal reference RPP30, and / or, The sum of the ratio of the peak height of the amplified product of exon 8 of SMN2 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 8 of SMN2 to the peak height of the amplified product of internal reference RPP30 was calculated. determining the copy number of SMN1 and / or SMN2 exon 7 and / or exon 8; By counting c.22dupA, c.683T / A and The peak height of the position was determined to determine c.22dupA, c.683T / A and Point mutation situation.

[0014] According to the present invention, The nucleotide sequence of the internal reference primer HBB is shown in SEQ ID No.13 and SEQ ID No.14; The nucleotide sequence of the internal reference primer RPP30 is shown in SEQ ID No. 15 and SEQ ID No. 16; The nucleotide sequences of primer combination 1 for specifically amplifying SMN1 and SMN2 that distinguish exon 7 sites are shown as SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3; The nucleotide sequences of primer combination 2 for specific amplification distinguishing SMN1 and SMN2 at exon 8 sites are shown in SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6; The nucleotide sequences of primer combination 3 for specific amplification of the c.22dupA containing mutation site are shown in SEQ ID No. 7 and SEQ ID No. 8; The nucleotide sequences of primer combination 4 for specific amplification of the c.683T / A-containing mutation site are shown in SEQ ID No. 9 and SEQ ID No. 10; Used for specific amplification of mutation sites The nucleotide sequences of primer combination 5 are shown as SEQ ID No.11 and SEQ ID No.12.

[0015] According to the present invention, the amplification reaction system: The final concentrations of dNTP and dUTP range from 100 μM to 400 μM, the final concentration of amplification primers range from 40 nM to 400 nM, the amount of DNA polymerase added ranges from 1 to 3 U, and the amount of UDG enzyme added ranges from 0.01 to 0.2 U.

[0016] Furthermore, the amplification reaction system: ddH2O, 2.045μL; 2.5X Multi PCR Buffer, 6μL; 10mM dGTP, dCTP, dATP, 1.35μL; 10 mM dTTP, 0.15 μL; 10 mM dUTP, 0.3 μL; 10μM SEQ ID NO. 1, 0.315μL; 10μM SEQ ID NO. 2, 0.165μL; 10μM SEQ ID NO. 3, 0.21μL; 10μM SEQ ID NO. 4, 0.315μL; 10μM SEQ ID NO. 5, 0.315μL; 10μM SEQ ID NO. 6, 0.315μL; 10μM SEQ ID NO. 7, 0.165μL; 10μM SEQ ID NO. 8, 0.165μL; 10μM SEQ ID NO. 9, 0.165μL; 10μM SEQ ID NO. 10, 0.165μL; 10μM SEQ ID NO. 11, 0.165μL; 10μM SEQ ID NO. 12, 0.165μL; 10μM SEQ ID NO. 13, 0.105μL; 10μM SEQ ID NO. 14, 0.105μL; 10μM SEQ ID NO. 15, 0.105μL; 10μM SEQ ID NO. 16, 0.105μL; 10μM SEQ ID NO. 17, 0.315μL; 10μM SEQ ID NO. 18, 0.315μL; DNA polymerase (5 U / μl), 0.45 μL; UDG enzyme (2 U / μl), 0.03 μL; DNA 1μl.

[0017] According to the present invention, the amplification conditions are: 50°C 2min 1cycle, 95°C 2min 1cycle, followed by 95°C 15sec, 56°C 30sec, 72°C 1min for a total of 25 cycles, and finally 72°C 30min.

[0018] As a second aspect of the present invention, a kit for detecting the human motor neuron survival gene SMN comprises: Universal Primer 1 and Universal Primer 2, Internal reference primers, including internal reference primer HBB; and / or, internal reference primer RPP30; Primer combination 1 for specifically amplifying SMN1 and SMN2 at exon 7 sites, including primers that can amplify the SMN1 gene in the genome but not the SMN2 gene and primers that can amplify the SMN2 gene in the genome but not the SMN1 gene, and the detection result can distinguish the SMN1 amplification product and the SMN2 amplification product at the exon 7 site; Primer combination 2 for specifically amplifying and distinguishing SMN1 and SMN2 at exon 8 loci, including primers capable of amplifying the SMN1 gene in the genome but not the SMN2 gene and primers capable of amplifying the SMN2 gene in the genome but not the SMN1 gene, and the detection result can distinguish the SMN1 amplification product and the SMN2 amplification product at the exon 8 loci; Primer combination 3 for specific amplification of the mutant template containing the mutation site c.22dupA; Primer combination 4 for specific amplification of the mutant template containing the mutation site c.683T / A; Used for specific amplification of mutation sites Primer combination 5 was used to specifically amplify the mutant template.

[0019] According to the present invention, the primer sequences of the universal primer 1 and the universal primer 2 are shown as SEQ ID No. 17 and SEQ ID No. 18, respectively.

[0020] According to the present invention, in the primer combination 1 for specifically amplifying SMN1 and SMN2 that distinguish exon 7 sites, in order to improve the discrimination and specificity of the primers, 2 to 3 differential bases are introduced at the 3' end of the primer that can amplify the SMN1 gene in the genome but not the SMN2 gene, and 2 to 3 differential bases are introduced at the 3' end of the primer that can amplify the SMN2 gene in the genome but not the SMN1 gene. At the same time, 5-6 base sequences that are completely different from the SMN gene are introduced at the 5' end of the specific primer that amplifies the SMN2 gene or the SMN1 gene. On the premise of ensuring that the two gene fragments are completely distinguished, the specificity of the primers is also ensured.

[0021] According to the present invention, in the primer combination 1 for specifically amplifying SMN1 and SMN2 that distinguish exon 8 sites, in order to improve the discrimination and specificity of the primers, 2 to 3 differential bases are introduced at the 3' end of the primer that can amplify the SMN1 gene in the genome but not the SMN2 gene, and 2 to 3 differential bases are introduced at the 3' end of the primer that can amplify the SMN2 gene in the genome but not the SMN1 gene. At the same time, 5-6 base sequences that are completely different from the SMN gene are introduced at the 5' end of the specific primer that amplifies the SMN2 gene or the SMN1 gene. On the premise of ensuring that the two gene fragments are completely distinguished, the specificity of the primers is also ensured.

[0022] According to the present invention, primer combination 3 for specific amplification of c.22dupA containing mutation site introduces 2-3 differential bases at the 3' end of the specific upstream primer, and only specifically amplifies the mutant template but not the wild template, thus ensuring the accuracy of the result.

[0023] According to the present invention, primer combination 4 for specific amplification of the mutation site c.683T / A introduces 2 to 3 differential bases at the 3' end of the specific upstream primer, and only specifically amplifies the mutant template but not the wild template, thus ensuring the accuracy of the result.

[0024] According to the present invention, for specific amplification of mutation sites The primer combination 5 introduced 2 to 3 differential bases at the 3' end of the specific upstream primer, which only specifically amplified the mutant template but not the wild template, ensuring the accuracy of the results.

[0025] According to the present invention, the nucleotide sequences of the primer combination 1 for specifically amplifying SMN1 and SMN2 that distinguish the exon 7 site are shown as SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3.

[0026] According to the present invention, the nucleotide sequences of the primer combination 2 for specifically amplifying SMN1 and SMN2 that distinguish the exon 8 site are shown as SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6.

[0027] According to the present invention, the nucleotide sequences of the primer combination 3 for specifically amplifying the gene containing the mutation site c.22dupA are shown in SEQ ID No.7 and SEQ ID No.8.

[0028] According to the present invention, the nucleotide sequences of the primer combination 4 for specifically amplifying the gene containing the mutation site c.683T / A are shown in SEQ ID No.9 and SEQ ID No.10.

[0029] According to the present invention, the method for specifically amplifying a mutation site The nucleotide sequences of primer combination 5 are shown in SEQ ID No.11 and SEQ ID No.12.

[0030] According to the present invention, the nucleotide sequence of the internal reference gene HBB is shown in SEQ ID No. 13 and SEQ ID No. 14.

[0031] According to the present invention, the nucleotide sequence of the internal reference gene RPP30 is shown in SEQ ID No. 15 and SEQ ID No. 16.

[0032] As a third aspect of the present invention, a calculation method for detecting the copy number variation of the human motor neuron survival gene SMN is used, using the kit as described in any one of the above or the method for detecting the human motor neuron survival gene SMN as described in any one of the above. By calculating the ratio of the peak height of the amplified product of exon 7 or exon 8 of SMN1 or SMN2 to the peak height of the amplified product of internal reference HBB, or, By calculating the ratio of the peak height of the amplified product of exon 7 or exon 8 of SMN1 or SMN2 to the peak height of the amplified product of the internal reference RPP30, or, By calculating the ratio of the peak height of the amplified product of exon 7 or exon 8 of SMN1 or SMN2 to the sum of the peak height of the amplified product of the internal reference HBB and the peak height of the amplified product of the internal reference RPP30, or, By calculating the sum of the ratio of the peak height of the amplified product of exon 7 of SMN1 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 7 of SMN1 to the peak height of the amplified product of internal reference RPP30, and / or, By calculating the sum of the ratio of the peak height of the amplified product of exon 8 of SMN1 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 8 of SMN1 to the peak height of the amplified product of internal reference RPP30, and / or, By calculating the sum of the ratio of the peak height of the amplified product of exon 7 of SMN2 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 7 of SMN2 to the peak height of the amplified product of internal reference RPP30, and / or, The sum of the ratio of the peak height of the amplified product of exon 8 of SMN2 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 8 of SMN2 to the peak height of the amplified product of internal reference RPP30 was calculated. determining the copy number of SMN1 and / or SMN2 exon 7 and / or exon 8; By counting c.22dupA, c.683T / A and The peak height of the position was determined to determine c.22dupA, c.683T / A and Point mutation situation.

[0033] According to the present invention, the calculation formula of the calculation method is: .

[0034] As a fourth aspect of the present invention, the above-mentioned calculation method is used in the accurate quantitative detection of high copy number of human motor neuron survival gene SMN.

[0035] Furthermore, the above-mentioned calculation method is used in the accurate quantitative detection of human motor neuron survival gene SMN with a copy number greater than 4.

[0036] The advantages of the present invention are: 1. The present invention has developed a method that can simultaneously detect SMN1 and SMN2 copy number variations, SMN1 minor variations, compound heterozygous mutations and silent carriers in one tube.

[0037] In the same reaction, in addition to detecting CNV and SNP of the SMN1 gene, it is also necessary to simultaneously detect CNV of the SMN2 gene, and the homology between the SMN1 and SMN2 genes is extremely high, with only a difference of 5 bases. Therefore, the present invention introduces 2 to 3 differential bases at the 3' end of the specific primer through clever primer design, thereby ensuring both the amplification efficiency and the specificity of the primer. In addition, based on the principle that capillary electrophoresis needs to distinguish different products by fragment size, although capillary electrophoresis can distinguish fragment differences of 1 base size, in order to avoid the risk of overlap of SMN1 and SMN2 fragments due to single-base insertion and deletion in the amplified region, 5 to 6 base sequences that are completely different from the SMN gene are introduced at the 5' end of the specific primer, thereby ensuring the specificity of the primer while ensuring complete distinction between the two gene fragments.

[0038] 2. Provides a convenient detection method for [2+0] type silent carrier mutations Due to the polymorphic sites of SMN1 gene Closely associated with [2+0] silent carriers, that is, when the subject has 2 copies of SMN1 and carries When the SMN1 gene is a point mutation, the subject is a [2+0] silent carrier. Based on this, the present invention has developed a method based on fluorescence PCR-capillary electrophoresis to simultaneously detect the SMN1 copy number and polymorphic sites in the same system. It does not require complex detection methods such as MLPA combined with NGS, nor does it require the introduction of multiple molecular genetic markers that are not clearly supported by research. It not only simplifies the detection process, but also simplifies the complexity of primers in the multiple amplification system, thereby ensuring the accuracy of the results.

[0039] 3. Provides a new method for calculating copy number variation Both fluorescent quantitative PCR and fluorescent PCR-capillary electrophoresis technology are based on PCR technology, and the advantage of PCR technology is that it is highly sensitive. It is precisely because of multiple cycles of exponential amplification that the initial differences can be highly amplified, which also brings great challenges to the accurate quantification of copy number. The present invention sets two internal references, which not only correct the errors in the sample addition process, but also play a role in complementary correction, and the reference substance is the key to correcting the differences between plates. The selection of the two, especially the selection of the internal reference and the calculation method, plays a very important role in the accuracy of the final result. The present invention uses two internal references to count the peak height values ​​of each target gene, and through a new algorithm, the Q value of each gene is calculated according to the following formula, and the copy number of the corresponding gene can be judged according to the Q value: .

[0040] 4. Provides a detection method that can accurately quantify higher copy numbers The published technology cannot distinguish SMN1 / 2 copy numbers greater than 4 copies, and can only generally classify ≥4 as a group. The present invention, by means of reaction system settings and improved calculation methods, can well distinguish between 4 copies and 5 copies of the gene.

[0041] 5. The selection of internal references and the unique calculation method achieve good distinction between 0-5 copies: The selection of dual internal references plays a role of complementary correction, solving the problem of poor high-copy distinction under a single internal reference system; and the calculation method of dual internal references is also different from the commonly used calculation method of taking the ratio of the target and the sum of two internal references. The present invention adopts a calculation method of calculating the two internal references separately and then taking the sum, which can well solve the problem that the deviation of the calculation result is amplified due to the abnormal result of one of the internal references, thereby leading to poor distinction. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Box plot of the calculation results for the internal reference 1 of SMN1-E7.

[0043] Figure 2 Box plot of the calculation results for the internal reference 1 of SMN2-E7.

[0044] Figure 3 Box plot of the calculation results for the internal reference 1 of SMN1-E8.

[0045] Figure 4 Box plot of the calculation results for the internal reference 1 of SMN2-E8.

[0046] Figure 5 Box plot of the calculation results of internal reference 2 for SMN1-E7.

[0047] Figure 6 Box plot of the calculation results of internal reference 2 for SMN2-E7.

[0048] Figure 7 Box plot of the calculation results of internal reference 2 for SMN1-E8.

[0049] Figure 8 Box plot of the calculation results of internal reference 2 of SMN2-E8.

[0050] Fig. 9 The ratio of SMN1-E7 to the sum of two internal references is shown in the box plot of the calculation results.

[0051] Fig.10 The ratio of SMN2-E7 to the sum of two internal references is shown in the box plot of the calculation results.

[0052] Fig.11It is the ratio of SMN1-E8 to the sum of two internal references, and the box plot of the calculation results is shown.

[0053] Fig.12 The ratio of SMN2-E8 to the sum of two internal references is shown in the box plot of the calculation results.

[0054] Fig.13 The box plot is the sum of the ratio of SMN1-E7 to internal reference 1 and the ratio of SMN1-E7 to internal reference 2.

[0055] Fig.14 The box plot is the sum of the ratio of SMN2-E7 to internal reference 1 and the ratio of SMN2-E7 to internal reference 2.

[0056] Fig.15 The box plot is the sum of the ratio of SMN1-E8 to internal reference 1 and the ratio of SMN1-E8 to internal reference 2.

[0057] Fig.16 The box plot is the sum of the ratio of SMN2-E8 to internal reference 1 and the ratio of SMN2-E8 to internal reference 2.

[0058] Figure 17-24 The sample electrophoresis result is shown in Figure 2. Fig.17 / 19 / 21 / 23 is the detection peak diagram using the method of Mode 3 of Example 2; Fig.18 / 20 / 22 / 24 is the peak graph of MLPA detection. Among them, Fig.17 The genes (or loci) represented by the numbers in the figure of / 19 / 21 / 23 are: 1: c.22dupA; 2: c.683T / A; 3: HBB; 4: SMN1-E7; 5: SMN2-E7; 6: SMN1-E8; 7: SMN2-E8; 8: ;9:RPP30. DETAILED DESCRIPTION

[0059] The present invention will be further described below in conjunction with specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0060] 1. Design of specific primers (1) SMN1 exon 7 and SMN2 exon 7 have only one base difference at the functional site, namely c.840C / T. ​​In order to distinguish the SMN1 gene and SMN2 gene in exon 7 by this site, the designed primers include: the upstream primer is the universal primer SEQ ID NO.1 for exon 7, and there are two specific downstream primers: one is used to amplify SMN1 but not SMN2, the sequence is shown in SEQ ID NO.2, and the other is used to amplify SMN2 but not SMN1, the sequence is shown in SEQ ID NO.3. In addition to the last base used to distinguish SMN1 and SMN2, these two primers introduce 2 to 3 different bases at the 3' end to further improve the discrimination and specificity of the primers.

[0061] (2) Exon 8 of SMN1 and exon 8 of SMN2 The differential site is the main distinguishing point, and the design principle is similar to that of exon 7, where the downstream is the universal primer SEQ ID NO. 4, and there are two upstream primers: one is used to amplify SMN1 but not SMN2, and the sequence is shown in SEQ ID NO. 5, and the other is used to amplify SMN2 but not SMN1, and the sequence is shown in SEQ ID NO. 6. In addition to the last base used to distinguish SMN1 from SMN2, these two primers introduce 2 to 3 differential bases at the 3' end to further improve the distinguishability and specificity of the primers.

[0062] (3) In order to distinguish SMN1 and SMN2 fragments in capillary electrophoresis results and avoid the risk of overlap of SMN1 and SMN2 fragments due to single-base insertions and deletions in the amplified region, 5-6 base sequences that are completely different from the SMN gene were introduced at the 5' end of the specific primer for amplifying the SMN2 gene. This ensured the specificity of the primer while ensuring complete distinction between the two gene fragments.

[0063] (4) High-frequency mutation sites in the Chinese population (c.22dupA, c.683T / A) and sites used to distinguish [2+0] silent carriers ( ) include: primer sequences for amplifying c.22dupA site as shown in SEQ ID NO.7 and SEQ ID NO.8, primer sequences for amplifying c.683T / A site as shown in SEQ ID NO.9 and SEQ ID NO.10, The primer sequences of the site are shown in SEQ ID NO. 11 and SEQ ID NO. 12. These three primers all introduce 2 to 3 differential bases at the 3' end of the specific upstream primer and can only specifically amplify the mutant template but not the wild template.

[0064] (5) The selected internal reference genes are one or both of HBB (Gene ID: 3043) and RPP30 (Gene ID: 10556). The primer sequences for amplifying the internal reference gene HBB are shown in SEQ ID NO. 13 and SEQ ID NO. 14, and the primer sequences for amplifying the internal reference gene RPP30 are shown in SEQ ID NO. 15 and SEQ ID NO. 16.

[0065] (6) To achieve equivalent amplification of exon 7, exon 8 and the internal reference gene, a common sequence was introduced at the 5' end of the above universal primers (exon 7 upstream primer and exon 8 downstream primer), and another common sequence was introduced at the 5' end of the specific primer for amplification of the specific fragment. A set of universal primers was also designed for these two common sequences, wherein universal primer 1 was fluorescently labeled, and the sequence was shown in SEQ ID NO. 17, which was convenient for subsequent capillary electrophoresis fragment analysis, and the other universal primer 2 was a common primer, and the sequence was shown in SEQ ID NO. 18.

[0066] Example 1: Reaction system optimization 1. Primer screening The SMN1 exon 7, SMN1 exon 8, SMN2 exon 7, SMN2 exon 8, c.22dupA, c.683T / A, , internal reference HBB and RPP30 were tested. For each site, a combination with better specificity and amplification efficiency was selected as a backup. Then, one or more primer sets of other sites were gradually added to the system of one site until all 16 or 18 primers were in the same tube to detect the SMN gene copy number and key mutation sites at one time.

[0067] After a large number of experimental optimizations, the primer sequences finally screened out are shown in Table 1 (the underlined parts are public sequences).

[0068] Table 1 Primer sequences 2. Reaction system setup and optimization 1) Extraction of genomic DNA: Extraction of genomic DNA from cells or human peripheral blood; 2) Reaction system optimization: After a large number of tests on different combinations of final concentrations of each component, the final concentrations (or addition amounts) of each component in the reaction system were finally determined as follows: dGTP / dCTP / dATP final concentration is 300μM, dTTP final concentration is 100μM, dUTP final concentration is 200μM, amplification primer final concentration range is 40nM~400nM, DNA polymerase addition amount is 2.25U, and UDG enzyme addition amount is 0.06U. The reaction system is shown in Table 2. Prepare the reaction system, vortex mix and centrifuge instantly; 3) PCR amplification: Place the above amplification mixture on a PCR instrument at 50℃ for 2min per cycle, 95℃ for 2min per cycle, then 95℃ for 15sec, 56℃ for 30sec, 72℃ for 1min for a total of 25 cycles, and finally 72℃ for 30min. 4) Capillary electrophoresis detection of amplified products: Take 8.8μL HiDi (highly deionized formamide) and mix it with 0.2μL molecular weight internal standard NS500 (fragment distribution: 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, 500), then take 1μL of the PCR amplification product from the previous step, vortex mix and centrifuge instantly, put it into the PCR instrument at 95℃ for 3min, put it on ice for 3min immediately after the end, and then detect it on the instrument; 5) Import the test data into GeneMapper software, select the preset analysis method and molecular weight internal standard parameters, and obtain the peak height value of each gene corresponding fragment; 6) Judgment of punctate sites: Whether a corresponding mutation occurs can be determined based on whether there is a product peak at the expected fragment size position of each punctate.

[0069] Table 2 Reaction system 3. Copy number calculation: ① The peak height ratio of exon 7 or exon 8 of the SMN gene of the test sample to the internal reference gene HBB of the test sample divided by the peak height ratio of exon 7 or exon 8 of the SMN gene of the control sample to the internal reference gene HBB of the control sample; ② The peak height ratio of exon 7 or exon 8 of the SMN gene of the test sample to the internal reference gene RPP30 of the test sample divided by the peak height ratio of exon 7 or exon 8 of the SMN gene of the control sample to the internal reference gene RPP30 of the control sample; ③ Add the above two results to get the Q value of the sample to be tested, and then determine the copy number of the corresponding gene based on the size of the Q value.

[0070] 4. Interpretation of results ①The SMN gene copy number is determined according to Table 3 Table 3 SMN gene copy number ② When the result calculated by the formula is within the above range, the copy number of the corresponding gene can be determined. If it is outside the range, it is recommended to re-test and then judge according to the above standards.

[0071] Example 2: Comparison of the discriminability of different algorithms for sample copy number 45 clinical screening samples were collected, and all samples were confirmed by SMA detection gold standard kit, namely SALSA MLPA Probemix P060 SMA Carrier of MRC-Holland, Netherlands, covering different genotypes. The detection was carried out according to the system of Example 1, and then different calculation methods were used for calculation, and the discrimination of each result was analyzed, and it was found that: Method 1: Using a single internal reference for calculation has good discrimination overall, but poor discrimination for samples with more than 4 copies (e.g. Figure 1-Figure 8 The specific results are as follows Figure 1-Figure 8 shown.

[0072] Method 2: Use two internal references and take the ratio of the target peak height to the sum of the two internal reference peak heights. The overall discrimination is better, but it is also unable to distinguish samples with ≥ 4 copies (e.g. Figure 9-12 The specific results are as follows Figure 9-12 shown.

[0073] .

[0074] Method 3: Using the calculation method provided by the present invention, there is a good distinction between samples with different copy numbers, especially for the case where the existing PCR method cannot distinguish more than 4 copy numbers, the calculation method of the present invention can achieve a good distinction between 0-5 copy number genes. The specific results are as follows Figure 13-16 shown.

[0075] .

[0076] Example 3: Method Validation 1. 48 EDTA-anticoagulated peripheral blood samples were collected for clinical screening, and the gold standard kit for SMA detection, namely SALSA MLPA Probemix P060 SMA Carrier from MRC-Holland of the Netherlands, was used as the reference method to verify the consistency of copy number detection results, and sequencing method was used to verify the consistency of point mutation results.

[0077] 2. The collected samples were used to extract genomic DNA using Jiachen Medical's automated nucleic acid extractor (HBH32B) and Meiji Bio's nucleic acid extraction or purification reagent (IVD3101-TL-06), and the concentration and purity were measured using a UV spectrophotometer. The concentration range was 10 ng / μl~80 ng / μl, and the purity A260 / A280 ratio was between 1.6 and 2.0.

[0078] 3. All samples were tested and analyzed according to Example 1. The copy number test results were consistent with the MLPA results. The samples with mutations detected in Example 1 all had corresponding mutations after sequencing verification, and the consistency reached 100%. The test results are shown in Table 4.

[0079] Table 4 Summary of test results of different clinical samples and MLPA copy number detection Electrophoresis results of some samples above: 1) Sample copy number relationship: SMN1-E7: SMN2-E7: SMN1-E8: SMN2-E8=1:2:1:2, c.22dupA, electrophoresis results are as follows Fig.17 and Fig.18 shown.

[0080] 2) Sample copy number relationship: SMN1-E7: SMN2-E7: SMN1-E8: SMN2-E8=2:1:2:1, c.683T / A, electrophoresis results are as follows Fig.19 and Fig. 20 shown.

[0081] 3) Sample copy number relationship: SMN1-E7: SMN2-E7: SMN1-E8: SMN2-E8=2:2:2:2. The electrophoresis results are as follows: Fig.21 and Fig. 22 shown.

[0082] 4) Sample copy number relationship and point mutation: SMN1-E7: SMN2-E7: SMN1-E8: SMN2-E8=4:1:5:0, The electrophoresis results are as follows Fig.23 and Fig.24 shown.

[0083] The results showed that under the system of the present invention, not only can the precise quantification of the copy numbers of SMN1 exon 7, SMN2 exon 7, SMN1 exon 8, and SMN2 exon 8 be completed in a single-tube reaction, but also higher copy numbers (such as 5 copies) can be distinguished, and point mutations can also be well detected.

[0084] In summary, the method for detecting the survival motor neuron gene SMN of the present invention ingeniously introduces 2 to 3 differential bases at the 3' end of the specific primer, which not only ensures the amplification efficiency of the primer, but also ensures the specificity of the primer; at the same time, 5 to 6 base sequences that are completely different from the SMN gene are introduced at the 5' end of the specific primer, which ensures the specificity of the primer while ensuring the complete distinction between the two gene fragments. At the same time, the method based on fluorescent PCR-capillary electrophoresis developed by the present invention simultaneously detects the SMN1 copy number and polymorphic sites in the same system. The method for distinguishing [2+0] silent carriers does not require complex detection methods such as MLPA combined with NGS, nor does it require the introduction of multiple molecular genetic markers that are not clearly supported by research. It simplifies the detection process and the complexity of primers in the multiplex amplification system, thereby ensuring the accuracy of the results. That is, the present invention realizes a method and application for simultaneously detecting the copy number of SMN1 and SMN2 and the minor variation of SMN1 in one tube.

[0085] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as The scope of protection of the present invention shall be based on the scope defined by the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting human motor neuron survival gene SMN, characterized in that: The primer combination 1 is capable of specifically amplifying SMN1 and SMN2 at the exon 7 site, the primer combination 2 is capable of specifically amplifying SMN1 and SMN2 at the exon 8 site, the primer combination 3 is capable of specifically amplifying the mutation site c.22dupA, the primer combination 4 is capable of specifically amplifying the mutation site c.683T / A, and the primer combination 5 is capable of specifically amplifying the mutation site c.683T / A. The primer combination 5 is used to amplify and obtain an amplified product; and amplifying with an internal reference primer HBB and / or an internal reference primer RPP30 to obtain an internal reference HBB amplification product and / or an internal reference RPP30 amplification product; By calculating the ratio of the peak height of the amplified product of exon 7 or exon 8 of SMN1 or SMN2 to the peak height of the amplified product of internal reference HBB, or, By calculating the ratio of the peak height of the amplified product of exon 7 or exon 8 of SMN1 or SMN2 to the peak height of the amplified product of the internal reference RPP30, or, By calculating the ratio of the peak height of the amplified product of exon 7 or exon 8 of SMN1 or SMN2 to the sum of the peak height of the amplified product of the internal reference HBB and the peak height of the amplified product of the internal reference RPP30, or, By calculating the sum of the ratio of the peak height of the amplified product of exon 7 of SMN1 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 7 of SMN1 to the peak height of the amplified product of internal reference RPP30, and / or, By calculating the sum of the ratio of the peak height of the amplified product of exon 8 of SMN1 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 8 of SMN1 to the peak height of the amplified product of internal reference RPP30, and / or, By calculating the sum of the ratio of the peak height of the amplified product of exon 7 of SMN2 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 7 of SMN2 to the peak height of the amplified product of internal reference RPP30, and / or, The sum of the ratio of the peak height of the amplified product of exon 8 of SMN2 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 8 of SMN2 to the peak height of the amplified product of internal reference RPP30 was calculated. determining the copy number of SMN1 and / or SMN2 exon 7 and / or exon 8; By counting c.22dupA, c.683T / A and The peak height of the position was determined to determine c.22dupA, c.683T / A and Point mutation situation.

2. The method for detecting the human motor neuron survival gene SMN according to claim 1, characterized in that: The nucleotide sequence of the internal reference primer HBB is shown in SEQ ID No.13 and SEQ ID No.14; The nucleotide sequence of the internal reference primer RPP30 is shown in SEQ ID No. 15 and SEQ ID No. 16; The nucleotide sequences of primer combination 1 for specifically amplifying SMN1 and SMN2 that distinguish exon 7 sites are shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3; The nucleotide sequences of primer combination 2 for specific amplification distinguishing SMN1 and SMN2 at exon 8 sites are shown in SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6; The nucleotide sequences of primer combination 3 for specific amplification of the c.22dupA containing mutation site are shown in SEQ ID No. 7 and SEQ ID No. 8; The nucleotide sequences of primer combination 4 for specific amplification of the c.683T / A-containing mutation site are shown in SEQ ID No. 9 and SEQ ID No. 10; Used to specifically amplify mutation sites The nucleotide sequences of primer combination 5 are shown in SEQ ID No.11 and SEQ ID No.

12.

3. The method for detecting the human motor neuron survival gene SMN according to claim 1 or 2, characterized in that: It is carried out in one tube, and the amplification reaction system is: The final concentrations of dNTP and dUTP range from 100 μM to 400 μM, the final concentration of amplification primers range from 40 nM to 400 nM, the amount of DNA polymerase added ranges from 1 to 3 U, and the amount of UDG enzyme added ranges from 0.01 to 0.2 U.

4. The method for detecting the human motor neuron survival gene SMN according to claim 1 or 2, characterized in that: It is carried out in one tube, and the amplification reaction system: ddH2O, 2.045μL; 2.5X Multi PCR Buffer, 6μL; 10mM dGTP, dCTP, dATP, 1.35μL; 10 mM dTTP, 0.15 μL; 10 mM dUTP, 0.3 μL; 10μM SEQ ID NO. 1, 0.315μL; 10μM SEQ ID NO. 2, 0.165μL; 10μM SEQ ID NO. 3, 0.21μL; 10μM SEQ ID NO. 4, 0.315μL; 10μM SEQ ID NO. 5, 0.315μL; 10μM SEQ ID NO. 6, 0.315μL; 10μM SEQ ID NO. 7, 0.165μL; 10μM SEQ ID NO. 8, 0.165μL; 10μM SEQ ID NO. 9, 0.165μL; 10μM SEQ ID NO. 10, 0.165μL; 10μM SEQ ID NO. 11, 0.165μL; 10μM SEQ ID NO. 12, 0.165μL; 10μM SEQ ID NO. 13, 0.105μL; 10μM SEQ ID NO. 14, 0.105μL; 10μM SEQ ID NO. 15, 0.105μL; 10μM SEQ ID NO. 16, 0.105μL; 10μM SEQ ID NO. 17, 0.315μL; 10μM SEQ ID NO. 18, 0.315μL; DNA polymerase (5 U / μl), 0.45 μL; UDG enzyme (2 U / μl), 0.03 μL; DNA 1 μL.

5. The method for detecting human motor neuron survival gene SMN according to claim 1 or 2, characterized in that: The amplification conditions were as follows: 50°C 2min 1cycle, 95°C 2min 1cycle, followed by 95°C 15sec, 56°C 30sec, 72°C 1min for a total of 25 cycles, and finally 72°C 30min.

6. A kit for detecting human motor neuron survival gene SMN, characterized in that: Include: Universal Primer 1 and Universal Primer 2, Internal reference primer, the internal reference primer HBB; and / or, internal reference primer RPP30; Primer combination 1 for specifically amplifying SMN1 and SMN2 that distinguish exon 7 sites, including primers that can amplify the SMN1 gene in the genome but not the SMN2 gene and primers that can amplify the SMN2 gene in the genome but not the SMN1 gene; Primer combination 2 for specifically amplifying and distinguishing SMN1 and SMN2 at exon 8 loci, including primers capable of amplifying the SMN1 gene in the genome but not the SMN2 gene and primers capable of amplifying the SMN2 gene in the genome but not the SMN1 gene; Primer combination 3 for specific amplification of c.22dupA containing mutation site; Primer combination 4 for specific amplification of the c.683T / A mutation site; Used for specific amplification of mutation sites Primer combination 5.

7. A kit for detecting the human motor neuron survival gene SMN as claimed in claim 6, characterized in that: The primer sequences of the universal primer 1 and the universal primer 2 are shown as SEQ ID No. 17 and SEQ ID No. 18, respectively.

8. A kit for detecting the human motor neuron survival gene SMN as claimed in claim 6, characterized in that: In the primer combination 1 for specifically amplifying SMN1 and SMN2 that distinguish the exon 7 site, 2 to 3 differential bases are introduced at the 3' end of the primer that can amplify the SMN1 gene in the genome but not the SMN2 gene, and 2 to 3 differential bases are introduced at the 3' end of the primer that can amplify the SMN2 gene in the genome but not the SMN1 gene, and at the same time, 5 to 6 base sequences that are completely different from the SMN gene are introduced at the 5' end of the specific primer that amplifies the SMN2 gene or the SMN1 gene; In the primer combination 1 for specifically amplifying SMN1 and SMN2 that distinguish the exon 8 site, 2 to 3 differential bases are introduced at the 3' end of the primer that can amplify the SMN1 gene in the genome but not the SMN2 gene, and 2 to 3 differential bases are introduced at the 3' end of the primer that can amplify the SMN2 gene in the genome but not the SMN1 gene, and at the same time, 5 to 6 base sequences that are completely different from the SMN gene are introduced at the 5' end of the specific primer that amplifies the SMN2 gene or the SMN1 gene; Primer combination 3, used for specific amplification of the c.22dupA containing mutation site, introduced 2 to 3 differential bases at the 3' end of the specific upstream primer; Primer combination 4, used for specific amplification of the c.683T / A-containing mutation site, introduced 2 to 3 differential bases at the 3' end of the specific upstream primer; Used to specifically amplify mutation sites The primer combination 5 introduced 2 to 3 differential bases at the 3' end of the specific upstream primer.

9. A kit for detecting the human motor neuron survival gene SMN as claimed in claim 6 or 7, characterized in that: The nucleotide sequences of the primer combination 1 for specifically amplifying SMN1 and SMN2 that distinguish the exon 7 site are shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3; The nucleotide sequences of the primer combination 2 for specifically amplifying SMN1 and SMN2 that distinguish the exon 8 site are shown in SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6; The nucleotide sequences of the primer combination 3 for specifically amplifying the c.22dupA-containing mutation site are shown in SEQ ID No. 7 and SEQ ID No. 8; The nucleotide sequences of the primer combination 4 for specific amplification of the c.683T / A-containing mutation site are shown in SEQ ID No. 9 and SEQ ID No. 10; The method is used to specifically amplify the mutation site The nucleotide sequences of primer combination 5 are shown in SEQ ID No. 11 and SEQ ID No. 12; The nucleotide sequence of the internal reference gene HBB is shown in SEQ ID No. 13 and SEQ ID No. 14; The nucleotide sequence of the internal reference gene RPP30 is shown in SEQ ID No. 15 and SEQ ID No.

16.

10. A calculation method for detecting the copy number variation of the human motor neuron survival gene SMN, characterized in that: The method comprises using the kit as described in any one of claims 6 to 9 or the method for detecting the human motor neuron survival gene SMN as described in any one of claims 1 to 5 for detection. By calculating the ratio of the peak height of the amplified product of exon 7 or exon 8 of SMN1 or SMN2 to the peak height of the amplified product of internal reference HBB, or, By calculating the ratio of the peak height of the amplified product of exon 7 or exon 8 of SMN1 or SMN2 to the peak height of the amplified product of the internal reference RPP30, or, By calculating the ratio of the peak height of the amplified product of exon 7 or exon 8 of SMN1 or SMN2 to the sum of the peak height of the amplified product of the internal reference HBB and the peak height of the amplified product of the internal reference RPP30, or, By calculating the sum of the ratio of the peak height of the amplified product of exon 7 of SMN1 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 7 of SMN1 to the peak height of the amplified product of internal reference RPP30, and / or, By calculating the sum of the ratio of the peak height of the amplified product of exon 8 of SMN1 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 8 of SMN1 to the peak height of the amplified product of internal reference RPP30, and / or, By calculating the sum of the ratio of the peak height of the amplified product of exon 7 of SMN2 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 7 of SMN2 to the peak height of the amplified product of internal reference RPP30, and / or, The sum of the ratio of the peak height of the amplified product of exon 8 of SMN2 to the peak height of the amplified product of internal reference HBB and the ratio of the peak height of the amplified product of exon 8 of SMN2 to the peak height of the amplified product of internal reference RPP30 was calculated. determining the copy number of SMN1 and / or SMN2 exon 7 and / or exon 8; By counting c.22dupA, c.683T / A and The peak height of the position was determined to determine c.22dupA, c.683T / A and Point mutation situation.

11. The calculation method for detecting the copy number variation of the survival motor neuron gene SMN according to claim 10, characterized in that: The calculation formula is: 。 12. An application of the calculation method as claimed in claim 10 in accurately and quantitatively detecting the high copy number of the human motor neuron survival gene SMN.

13. An application of the calculation method as claimed in claim 10 in the accurate quantitative detection of the human motor neuron survival gene SMN with a copy number greater than 4.

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