A method for detecting human motor neuron survival gene SMN

Abstract

The present invention discloses a method for detecting the human survival motor neuron gene (SMN), a kit for detecting the human survival motor neuron gene (SMN), a method for calculating the copy number variation of the human survival motor neuron gene (SMN), and the application of the calculation method for accurately and quantitatively detecting high copy numbers of the human survival motor neuron gene (SMN). The method of the present invention enables simultaneous detection of copy number variations of SMN1 exon 7 and exon 8 and SMN2 exon 7 and exon 8, SMN1 minor mutations, compound heterozygous mutations, and silent carriers in a single tube.

Description

Technical Field

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

[0002] Spinal muscular atrophy (SMA) is the most common autosomal recessive neuromuscular disease in children, characterized by muscle weakness and atrophy caused by degeneration of α-motor neurons in the anterior horn of the spinal cord. The incidence of SMA is approximately 1 in 10,000, and the carrier rate in Asian populations is approximately 1 in 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 modifying gene SMN2. These two genes are highly homologous and have inverted repeats. This characteristic 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 development 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 SMN1 gene expression. Currently, there are two mutation sites unique to Chinese people found in large-scale 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] SMA patients, carriers, and normal individuals can be distinguished based on the number of SMN1 copies. A subject with zero SMN1 copies is classified as an SMA patient; a subject with one SMN1 copy is classified as an SMA carrier. If they also carry a minor mutation, they are considered compound heterozygous. A subject with two SMN1 copies, carrying one copy on each chromosome, is classified as a "1+1" type, representing a normal individual. However, a special type of carrier (approximately 4% of carriers) has both copies on the same chromosome, while the SMN1 gene is missing from the other chromosome. This type, known as a "2+0" type, is termed a silent carrier. There is a 50% chance that the SMN1-free chromosome will be passed on to offspring, resulting in SMA. Early strategies for detecting the "2+0" type involved family analysis using several molecular genetic markers. This approach is cumbersome and unsuitable for screening. Furthermore, such a system requires more complex primers, which can affect amplification stability. There are 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] Approximately 80-90% of the protein produced by the SMN2 modifier gene is truncated and unstable, while an additional 10-20% of the full-length protein is functional, partially compensating for the loss of SMN1 gene function caused by the mutation. According to international and domestic management consensus, SMA is generally classified into types I-IV based on the patient's SMN2 copy number, age of onset, and demonstrated motor ability. SMN2 copy number increases with each level, while the age of onset and severity of the disease decrease. Therefore, SMN2 copy number is often used as an important reference data in clinical trials of drugs targeting SMN protein regulation as a therapeutic strategy, and in newborn screening as a key biomarker for pre-symptomatic treatment assessment.

[0007] This suggests that, in addition to measuring SMN1 gene copy number, mutations at key SMN1 gene sites are extremely valuable for screening healthy individuals. Accurately quantifying SMN2 gene copy number also provides valuable guidance for clinicians in developing treatment plans for individual patients. A rapid and convenient testing technology or product that can simultaneously meet these needs is clinically needed.

[0008] Currently, most SMA mutation detection technologies reported and marketed domestically and internationally rely on real-time quantitative PCR (PCR). This technology has a short detection cycle and widespread instrument penetration, making it suitable for population screening. However, it does not detect hotspot mutation sites, which can lead to missed detection of patients with compound heterozygous mutations or [2+0] silent carriers, causing irreparable harm to patients and their families. Furthermore, it cannot accurately quantify copy numbers ≥2. Detecting SMN1 and SMN2 copy numbers requires separate, multi-tube reactions, which is complex and inconvenient. Multiplex ligation-dependent probe amplification (MLPA) based on a capillary electrophoresis platform is the current consensus-recommended gold standard for detecting SMN1 and SMN2 copy number variations. It not only accurately quantifies copy number but also detects point mutations. However, this method is expensive, cumbersome (requiring denaturation, hybridization, ligation, amplification, and on-device testing), and time-consuming (the entire process takes two days), making it unsuitable for large-scale screening.

[0009] Fluorescence PCR-capillary electrophoresis uses fluorescent dye-labeled primers to run the amplified PCR products through electrophoresis in capillary channels, with software used to collect images and analyze the size of the amplified fragments. The earliest attempt to use this technology to simultaneously detect SMN copy number and mutations in each exon was patented (US7875432B2). Compared to other detection technologies, this method has the advantages of relatively simple operation and a short detection cycle. However, while it can simultaneously detect copy number and point mutations, it requires two separate amplification systems and can only determine the ratio of SMN1 to SMN2 copy numbers, failing to accurately quantify SMN1 and SMN2 copy numbers. However, this attempt made it possible to detect SMN genes using fluorescence PCR-capillary electrophoresis. Later, an improved method for SMN detection 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 the 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] To address the shortcomings of current methods for detecting SMA-related gene copy number, compound heterozygous mutations, and silent carriers, the present invention screened and compared a large number of alternative internal references, ultimately selecting one or both of the hemoglobin subunit beta gene (HBB) and ribonuclease P / MRP subunit p30 (RPP30) as internal references for calculation of the results. Furthermore, through extensive primer screening and system optimization, this method achieves simultaneous amplification of SMN1 and SMN2 exons 7 and 8, two high-frequency mutation sites in the Chinese population, and a key mutation site for distinguishing [2+0] silent carriers in a single tube. This system design enables high-throughput detection of multiple targets at a lower cost, better meeting clinical needs.

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

[0013] To achieve the above object, the present invention adopts the following technical solutions:

[0014] As a first aspect of the present invention, a method for detecting the human survival motor neuron gene SMN is provided, which comprises a primer combination 1 capable of specifically amplifying SMN1 and SMN2 at the site of exon 7, a primer combination 2 capable of specifically amplifying SMN1 and SMN2 at the site of exon 8, a primer combination 3 capable of specifically amplifying a site containing the mutation site c.22dupA, a primer combination 4 capable of specifically amplifying a site containing the mutation site c.683T / A, and a primer combination 5 capable of specifically amplifying a site containing the mutation site The primer combination 5 is used for amplification to obtain an amplified product;

[0015] 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;

[0016] 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,

[0017] 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 RPP30, or,

[0018] 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 internal reference HBB and the peak height of the amplified product of internal reference RPP30, or,

[0019] 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,

[0020] 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,

[0021] 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,

[0022] The sum of the ratio of the peak height of the amplified product of exon 8 of SMN2 to the peak height of the internal reference HBB amplified product and the ratio of the peak height of the amplified product of exon 8 of SMN2 to the peak height of the internal reference RPP30 amplified product was calculated.

[0023] determining the copy number of SMN1 and / or SMN2 exon 7 and / or exon 8;

[0024] 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.

[0025] According to the present invention,

[0026] The nucleotide sequences of the internal reference primer HBB are shown in SEQ ID No. 13 and SEQ ID No. 14;

[0027] The nucleotide sequences of the internal reference primer RPP30 are shown in SEQ ID No. 15 and SEQ ID No. 16;

[0028] 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;

[0029] The nucleotide sequences of primer combination 2 for specifically amplifying and distinguishing SMN1 and SMN2 at exon 8 sites are shown in SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6;

[0030] The nucleotide sequences of primer combination 3 for specifically amplifying the c.22dupA mutation site are shown in SEQ ID No. 7 and SEQ ID No. 8;

[0031] The nucleotide sequences of primer combination 4 for specifically amplifying the c.683T / A mutation site are shown in SEQ ID No. 9 and SEQ ID No. 10;

[0032] 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.

[0033] According to the present invention, the amplification reaction system:

[0034] 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.

[0035] Furthermore, the amplification reaction system:

[0036] ddH2O, 2.045μL;

[0037] 2.5X Multi PCR Buffer, 6μL;

[0038] 10mM dGTP, dCTP, dATP, 1.35μL;

[0039] 10 mM dTTP, 0.15 μL;

[0040] 10 mM dUTP, 0.3 μL;

[0041] 10μM SEQ ID NO. 1, 0.315μL;

[0042] 10μM SEQ ID NO. 2, 0.165μL;

[0043] 10 μM SEQ ID NO. 3, 0.21 μL;

[0044] 10μM SEQ ID NO. 4, 0.315μL;

[0045] 10μM SEQ ID NO. 5, 0.315μL;

[0046] 10μM SEQ ID NO. 6, 0.315μL;

[0047] 10μM SEQ ID NO. 7, 0.165μL;

[0048] 10μM SEQ ID NO. 8, 0.165μL;

[0049] 10μM SEQ ID NO. 9, 0.165μL;

[0050] 10μM SEQ ID NO. 10, 0.165μL;

[0051] 10μM SEQ ID NO. 11, 0.165μL;

[0052] 10μM SEQ ID NO. 12, 0.165μL;

[0053] 10μM SEQ ID NO. 13, 0.105μL;

[0054] 10μM SEQ ID NO. 14, 0.105μL;

[0055] 10μM SEQ ID NO. 15, 0.105μL;

[0056] 10μM SEQ ID NO. 16, 0.105μL;

[0057] 10μM SEQ ID NO. 17, 0.315μL;

[0058] 10μM SEQ ID NO. 18, 0.315μL;

[0059] DNA polymerase (5 U / μl), 0.45 μL;

[0060] UDG enzyme (2 U / μl), 0.03 μL;

[0061] DNA 1μl.

[0062] According to the present invention, the amplification conditions are: 50°C 2 min 1 cycle, 95°C 2 min 1 cycle, followed by 95°C 15 sec, 56°C 30 sec, 72°C 1 min for a total of 25 cycles, and finally 72°C 30 min.

[0063] As a second aspect of the present invention, a kit for detecting the human survival motor neuron gene SMN comprises:

[0064] Universal primer 1 and universal primer 2,

[0065] Internal reference primers, including internal reference primer HBB; and / or internal reference primer RPP30;

[0066] Primer combination 1 for specifically amplifying and distinguishing SMN1 and SMN2 at exon 7, 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. The test results can distinguish between the SMN1 amplification product and the SMN2 amplification product at exon 7;

[0067] Primer combination 2 for specifically amplifying and distinguishing SMN1 and SMN2 at exon 8, 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. The test results can distinguish between the SMN1 amplification product and the SMN2 amplification product at exon 8;

[0068] Primer combination 3 for specific amplification of the mutant template containing the mutation site c.22dupA;

[0069] Primer combination 4 for specific amplification of the mutant template containing the mutation site c.683T / A;

[0070] For specific amplification of mutation sites Primer combination 5 was used to specifically amplify the mutant template.

[0071] 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.

[0072] 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. This ensures the specificity of the primers while ensuring complete discrimination between the two gene fragments.

[0073] 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. This ensures the specificity of the primers while ensuring complete discrimination between the two gene fragments.

[0074] According to the present invention, primer combination 3 for specific amplification of the mutation site c.22dupA introduces 2 to 3 differential bases at the 3' end of the specific upstream primer, which specifically amplifies only the mutant template but not the wild-type template, thus ensuring the accuracy of the results.

[0075] 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, which specifically amplifies only the mutant template but not the wild template, ensuring the accuracy of the results.

[0076] According to the present invention, for specific amplification of the mutation site Primer combination 5 introduces 2 to 3 differential bases at the 3' end of the specific upstream primer, which specifically amplifies only the mutant template but not the wild-type template, ensuring the accuracy of the results.

[0077] According to the present invention, the nucleotide sequences of the 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.

[0078] According to the present invention, the nucleotide sequences of the primer combination 2 for specifically amplifying SMN1 and SMN2 that distinguish exon 8 sites are shown as SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6.

[0079] 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 as SEQ ID No. 7 and SEQ ID No. 8.

[0080] 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.

[0081] According to the present invention, the method for specifically amplifying the mutation site The nucleotide sequences of primer combination 5 are shown as SEQ ID No. 11 and SEQ ID No. 12.

[0082] 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.

[0083] 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.

[0084] As a third aspect of the present invention, a calculation method for detecting copy number variation of the human survival motor neuron gene SMN is performed using the kit described in any one of the above or the method for detecting the human survival motor neuron gene SMN described in any one of the above.

[0085] 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,

[0086] 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 RPP30, or,

[0087] 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 internal reference HBB and the peak height of the amplified product of internal reference RPP30, or,

[0088] 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,

[0089] 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,

[0090] 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,

[0091] The sum of the ratio of the peak height of the amplified product of exon 8 of SMN2 to the peak height of the internal reference HBB amplified product and the ratio of the peak height of the amplified product of exon 8 of SMN2 to the peak height of the internal reference RPP30 amplified product was calculated.

[0092] determining the copy number of SMN1 and / or SMN2 exon 7 and / or exon 8;

[0093] 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.

[0094] According to the present invention, the calculation formula of the calculation method is:

[0095] .

[0096] 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 the human motor neuron survival gene SMN.

[0097] 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.

[0098] The advantages of the present invention are:

[0099] 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.

[0100] Since in the same reaction, in addition to detecting CNV and SNP of the SMN1 gene, CNV of the SMN2 gene also needs to be detected simultaneously, and the SMN1 and SMN2 genes have extremely high homology, with only a difference of 5 bases, 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 SMN1 and SMN2 fragment overlap due to single-base insertions and deletions in the amplified region, 5-6 base sequences that are completely different from the SMN gene are introduced at the 5' end of the specific primer. While ensuring complete differentiation of the two gene fragments, the specificity of the primer is also ensured.

[0101] 2. Provides a convenient method for detecting [2+0] silent carrier mutations

[0102] Due to the polymorphic sites of the SMN1 gene Closely associated with [2+0] silent carriers, that is, when the subject has 2 copies of SMN1 and carries When a point mutation occurs, 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 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 simplifies the detection process and the complexity of primers in the multiplex amplification system, thereby ensuring the accuracy of the results.

[0103] 3. Provides a new method for calculating copy number variation

[0104] Whether it is fluorescent quantitative PCR or fluorescent PCR-capillary electrophoresis technology, both are based on PCR technology, and the advantage of PCR technology is that it is highly sensitive. And it is precisely because after multiple cycles of exponential amplification, the initial difference 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 error in the sample addition process, but also play a role in mutual correction, and the reference substance is the key to correcting the difference 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, calculates the Q value of each gene according to the following formula, and the copy number of the corresponding gene can be judged according to the Q value:

[0105] .

[0106] 4. Provides a detection method that can accurately quantify higher copy numbers

[0107] Published technologies cannot distinguish SMN1 / 2 copy numbers greater than 4 copies and can only generally classify ≥4 as a group. However, the present invention, with the help of improved reaction system settings and calculation methods, can effectively distinguish between 4 and 5 copies of the gene.

[0108] 5. The selection of internal references and a unique calculation method achieve good discrimination of 0-5 copies: The selection of two internal references plays a role of mutual complementation and correction, solving the problem of poor high-copy discrimination under the single internal reference system; and the calculation method of the two 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 effectively solve the problem of poor discrimination caused by the abnormal result of one of the internal references, which leads to the amplification of the calculation result deviation. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Figure 1 Box plot of the calculation results for the internal reference 1 of SMN1-E7.

[0110] Figure 2 Box plot of the calculation results for the internal reference 1 of SMN2-E7.

[0111] Figure 3 Box plot of the calculation results for the internal reference 1 of SMN1-E8.

[0112] Figure 4 Box plot of the calculation results for the internal reference 1 of SMN2-E8.

[0113] Figure 5 Box plot of the calculation results for internal reference 2 of SMN1-E7.

[0114] Figure 6 Box plot of the calculation results for internal reference 2 of SMN2-E7.

[0115] Figure 7 Box plot of the calculation results of internal reference 2 for SMN1-E8.

[0116] Figure 8 Box plot of the calculation results for the internal reference 2 of SMN2-E8.

[0117] Figure 9 The box plot is the ratio of SMN1-E7 to the sum of two internal references.

[0118] Figure 10 The ratio of SMN2-E7 to the sum of two internal references is shown in the box plot of the calculation results.

[0119] Figure 11 The box plot is the ratio of SMN1-E8 to the sum of two internal references.

[0120] Figure 12 The ratio of SMN2-E8 to the sum of two internal references is shown in the box plot of the calculation results.

[0121] Figure 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.

[0122] Figure 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.

[0123] Figure 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.

[0124] Figure 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.

[0125] Figure 17-24 The electrophoresis results of the samples are shown in Figure 2. Figure 17 / 19 / 21 / 23 are the detection peak graphs using the method of Mode 3 of Example 2; Figure 18 / 20 / 22 / 24 are the peak graphs of MLPA detection. Figure 17 The genes (or loci) represented by the numbers in the figures 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

[0126] 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 intended to illustrate the present invention only and are not intended to limit the scope of the invention.

[0127] 1. Design of specific primers

[0128] (1) SMN1 exon 7 and SMN2 exon 7 differ in their functional sites by only one base, c.840C / T. To distinguish SMN1 and SMN2 genes in exon 7 based on this site, the primers designed included: the upstream primer was the universal primer SEQ ID NO. 1 for exon 7, and two specific downstream primers: one was used to amplify SMN1 but not SMN2, and the sequence was shown in SEQ ID NO. 2; the other was used to amplify SMN2 but not SMN1, and the sequence was shown in SEQ ID NO. 3. In addition to the last base used to distinguish SMN1 from SMN2, these two primers introduced 2 to 3 different bases at the 3' end to further improve the discrimination and specificity of the primers.

[0129] (2) Exon 8 of SMN1 and exon 8 of SMN2 The differential site serves as the primary distinguishing point, and the design principle is similar to that of exon 7. The downstream primer is the universal primer SEQ ID NO. 4, and there are two upstream primers: one amplifies SMN1 but not SMN2, and the sequence is shown in SEQ ID NO. 5; the other amplifies 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 incorporate two to three differential bases at the 3' end to further enhance primer discrimination and specificity.

[0130] (3) In order to distinguish SMN1 and SMN2 fragments in capillary electrophoresis results and avoid the risk of SMN1 and SMN2 fragment overlap 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 the complete distinction between the two gene fragments.

[0131] (4) High-frequency mutation sites in the Chinese population (c.22dupA, c.683T / A) and sites used to distinguish [2+0] silent carriers ( ) primers 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, and primer sequences for amplifying The primer sequences of the sites 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.

[0132] (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 NOs. 13 and 14, and the primer sequences for amplifying the internal reference gene RPP30 are shown in SEQ ID NOs. 15 and 16.

[0133] (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 universal primers (exon 7 upstream primer and exon 8 downstream primer), and another common sequence was introduced at the 5' end of the specific primers for amplification of the specific fragments. A set of universal primers was also designed for these two common sequences. Universal primer 1 was fluorescently labeled, and its sequence was shown in SEQ ID NO. 17, which facilitated subsequent capillary electrophoresis fragment analysis. The other universal primer 2 was a common primer, and its sequence was shown in SEQ ID NO. 18.

[0134] Example 1: Reaction system optimization

[0135] 1. Primer Screening

[0136] 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 for 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.

[0137] After a large number of experimental optimizations, the primer sequences finally screened out are shown in Table 1 (the underlined parts are public sequences).

[0138] Table 1 Primer sequences

[0139]

[0140] 2. Reaction system setup and optimization

[0141] 1) Extraction of genomic DNA: Extraction of genomic DNA from cells or human peripheral blood;

[0142] 2) Reaction System Optimization: After extensive testing of various combinations of final component concentrations, the final concentrations (or added amounts) of the reaction system were determined as follows: dGTP / dCTP / dATP at 300 μM, dTTP at 100 μM, dUTP at 200 μM, amplification primers at a concentration range of 40 nM to 400 nM, DNA polymerase at 2.25 U, and UDG at 0.06 U. The reaction system is shown in Table 2. Prepare the reaction system, vortex to mix, and centrifuge briefly.

[0143] 3) PCR amplification: Place the above amplification mixture on a PCR instrument and cycle at 50°C for 2 minutes per cycle, then 95°C for 2 minutes per cycle, followed by 95°C for 15 seconds, 56°C for 30 seconds, and 72°C for 1 minute for a total of 25 cycles, and finally at 72°C for 30 minutes.

[0144] 4) Capillary electrophoresis detection of amplified products: Mix 8.8 μL of HiDi (highly deionized formamide) with 0.2 μL of molecular weight internal standard NS500 (fragment distribution: 75, 100, 139, 150, 160, 200, 250, 300, 340, 350, 400, 450, 490, 500). Then, take 1 μL of the PCR product from the previous step, vortex to mix, centrifuge briefly, and place in a PCR instrument at 95°C for 3 minutes. Immediately place on ice for 3 minutes before testing.

[0145] 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;

[0146] 6) Determination of punctate sites: Whether a corresponding mutation has occurred can be determined by whether there is a product peak at the expected fragment size position of each punctate site.

[0147] Table 2 Reaction system

[0148]

[0149] 3. Copy number calculation:

[0150] ① The peak height ratio of exon 7 or exon 8 of the SMN gene in the test sample to the internal reference gene HBB in the test sample is divided by the peak height ratio of exon 7 or exon 8 of the SMN gene in the control sample to the internal reference gene HBB in the control sample;

[0151] ② The peak height ratio of exon 7 or exon 8 of the SMN gene in the test sample to the internal reference gene RPP30 in the test sample divided by the peak height ratio of exon 7 or exon 8 of the SMN gene in the control sample to the internal reference gene RPP30 in the control sample;

[0152] ③ Add the above two results to obtain 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.

[0153] 4. Interpretation of results

[0154] ①The SMN gene copy number was determined according to Table 3

[0155] Table 3 SMN gene copy number

[0156]

[0157] 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 retest and then interpret it according to the above standards.

[0158] Example 2: Comparison of the discriminative performance of different algorithms on sample copy number

[0159] 45 clinical screening samples were collected and confirmed using the gold standard SMA detection kit, the SALSA MLPA Probemix P060 SMA Carrier from MRC-Holland, the Netherlands, to cover different genotypes. Testing was performed using the system described in Example 1, and different calculation methods were used. The discriminatory nature of the results was analyzed, revealing the following:

[0160] Method 1: Using a single internal reference for calculation has good discrimination overall, but it is not good for samples with more than 4 copies (e.g. Figures 1-8 The specific results are as follows Figures 1-8 shown.

[0161] Method 2: Using two internal references, taking 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. Figures 9-12 The specific results are as follows Figures 9-12 shown.

[0162] .

[0163] Method 3: Using the calculation method provided by the present invention, there is a good distinction between samples with different copy numbers. In particular, 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.

[0164] .

[0165] Example 3: Method Validation

[0166] 1. EDTA-anticoagulated peripheral blood samples were collected from 48 clinical screening patients. The gold standard SMA detection kit, the SALSA MLPA Probemix P060 SMA Carrier from MRC-Holland (The Netherlands), was used as a reference method to verify the consistency of copy number detection results. Sequencing was used to verify the consistency of point mutation results.

[0167] 2. Genomic DNA was extracted from the collected samples using a Jiachen Medical automated nucleic acid extractor (HBH32B) in combination with Meiji Biotech nucleic acid extraction or purification reagents (IVD3101-TL-06). The concentration and purity were measured using a UV spectrophotometer. The concentration range was 10 ng / μl to 80 ng / μl, and the purity A260 / A280 ratio was between 1.6 and 2.0.

[0168] 3. All samples were tested and analyzed according to Example 1. The copy number test results were consistent with the MLPA results. Sequencing confirmed the presence of the corresponding mutation in all samples detected in Example 1, with 100% consistency. The test results are shown in Table 4.

[0169] Table 4 Summary of test results of different clinical samples and MLPA copy number detection

[0170]

[0171]

[0172] Electrophoresis results of some of the above samples:

[0173] 1) Sample copy number relationship: SMN1-E7: SMN2-E7: SMN1-E8: SMN2-E8 = 1:2:1:2, c.22dupA, electrophoresis results are as follows Figure 17 and Figure 18 shown.

[0174] 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 Figure 19 and Figure 20 shown.

[0175] 3) Sample copy number relationship: SMN1-E7: SMN2-E7: SMN1-E8: SMN2-E8=2:2:2:2, electrophoresis results are as follows Figure 21 and Figure 22 shown.

[0176] 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 Figure 23 and Figure 24 shown.

[0177] The results showed that under the system of the present invention, not only can the copy numbers of SMN1 exon 7, SMN2 exon 7, SMN1 exon 8, and SMN2 exon 8 be accurately quantified in a single-tube reaction, but higher copy numbers (such as 5 copies) can also be distinguished, and point mutations can also be well detected.

[0178] In summary, the present invention provides a method for detecting the human survival motor neuron gene SMN, which cleverly introduces 2 to 3 differential bases at the 3' end of the specific primer, ensuring both the amplification efficiency and the specificity of the primer. At the same time, 5 to 6 base sequences completely different from the SMN gene are introduced at the 5' end of the specific primer, ensuring the complete differentiation of the two gene fragments while also ensuring the specificity of the primer. At the same time, the method developed by the present invention based on fluorescence PCR-capillary electrophoresis can simultaneously detect SMN1 copy number and polymorphic sites in the same system. This 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 have not been clearly supported by research. This simplifies the detection process and the primer complexity of the multiplex amplification system, thereby ensuring the accuracy of the results. This method and application achieves simultaneous detection of SMN1 and SMN2 copy numbers and SMN1 minor variants in a single tube.

[0179] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as

[0180] The scope of protection of the present invention shall be based on the scope defined by the claims. For those skilled in the art, it is possible to make several improvements and modifications without departing from the spirit and scope of the present invention, and such improvements and modifications shall also be considered 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 site containing the mutation c.22dupA, the primer combination 4 is capable of specifically amplifying the site containing the mutation c.683T / A, and the primer combination 5 is capable of specifically amplifying the site containing the mutation c. 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 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 internal reference HBB and the peak height of the amplified product of 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 internal reference HBB amplified product and the ratio of the peak height of the amplified product of exon 8 of SMN2 to the peak height of the internal reference RPP30 amplified product 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; The nucleotide sequences of the internal reference primer HBB are shown in SEQ ID No. 13 and SEQ ID No. 14; The nucleotide sequences of the internal reference primer RPP30 are 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 specifically amplifying and 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 specifically amplifying the c.22dupA mutation site are shown in SEQ ID No. 7 and SEQ ID No. 8; The nucleotide sequences of primer combination 4 for specifically amplifying the c.683T / A mutation site are shown in SEQ ID No. 9 and SEQ ID No. 10; 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.

2. The method for detecting the human survival motor neuron gene SMN according to claim 1, wherein: 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.

3. The method for detecting the human survival motor neuron gene SMN according to claim 1, wherein: It is carried out in one tube, and the amplification reaction system: ddH2O, 2.045μL; 2.5X Multi PCR Buffer, 6μL; 10mMdGTP, 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; 10 μM SEQ ID NO. 3, 0.21 μL; 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; 1 μL of DNA.

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

5. 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, comprising a primer capable of amplifying the SMN1 gene in the genome but not the SMN2 gene, and a primer capable of amplifying the SMN2 gene in the genome but not the SMN1 gene; Primer combination 2 for specifically amplifying and distinguishing SMN1 and SMN2 at the exon 8 locus, comprising a primer capable of amplifying the SMN1 gene in the genome but not the SMN2 gene, and a primer capable of amplifying the SMN2 gene in the genome but not the SMN1 gene; Primer combination 3 for specific amplification of the c.22dupA mutation site; Primer combination 4 for specific amplification of the c.683T / A mutation site; For specific amplification of mutation sites Primer combination 5; The nucleotide sequences of the 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 the primer combination 2 for specifically amplifying SMN1 and SMN2 that distinguish exon 8 sites 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 mutation site are shown in SEQ ID No. 7 and SEQ ID No. 8; 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; 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 sequences of the internal reference gene HBB are shown in SEQ ID No. 13 and SEQ ID No. 14; The nucleotide sequences of the internal reference gene RPP30 are shown in SEQ ID No. 15 and SEQ ID No.

16.

6. A kit for detecting the human survival motor neuron gene SMN according to claim 5, 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.

7. A calculation method for detecting copy number variation of the human survival motor neuron gene SMN for non-disease diagnosis purposes, characterized in that: The method for detecting the human survival motor neuron gene SMN is performed using the kit according to any one of claims 5 to 6 or the method for detecting the human survival motor neuron gene SMN according to any one of claims 1 to 4. 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 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 internal reference HBB and the peak height of the amplified product of 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 internal reference HBB amplified product and the ratio of the peak height of the amplified product of exon 8 of SMN2 to the peak height of the internal reference RPP30 amplified product 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.

8. The calculation method for detecting the copy number variation of the human survival motor neuron gene SMN for non-disease diagnosis purposes according to claim 7, characterized in that: The calculation formula is: 。 9. Use of the calculation method for non-disease diagnosis purposes as claimed in claim 7 in accurately and quantitatively detecting high copy numbers of the human survival motor neuron gene SMN.

10. Use of the calculation method for non-disease diagnosis purposes according to claim 7 in accurately and quantitatively detecting a copy number greater than 4 of the human survival motor neuron gene SMN.

Images