A primer probe set and kit for detecting the SMN1 pathogenic gene of spinal muscular atrophy

Abstract

The present invention discloses a primer probe set and a kit for detecting the SMN1 pathogenic gene of spinal muscular atrophy. The kit provided by the present invention comprises three kinds of gene detection liquids, wherein there are a plurality of primers and probes, and the primers and probes are shown in SEQ ID NO: 1 to SEQ ID NO: 33 respectively. The kit provided by the present invention overcomes the shortcomings and defects of the prior art, and uses the fluorescent probe melting curve technology to simultaneously detect the copy number and common point mutations of the SMN1 gene, and can detect trace samples including dried blood spots / saliva, and can simultaneously detect SMA patients and carriers, and has the advantages of short time consumption, simple operation, strong specificity, high sensitivity, high accuracy and low cost.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a primer probe set and a kit for detecting a spinal muscular atrophy pathogenic gene SMN1. Background Art

[0002] Spinal muscular atrophy (SMA) is the most common neuromuscular disease in children. Its clinical features are mainly muscle weakness and atrophy caused by degeneration of α-motor neurons in the anterior horn of the spinal cord. The clinical symptoms of SMA patients vary greatly, and the disease can occur from birth to adulthood. The main manifestations are progressive muscle weakness and atrophy mainly in the proximal limbs. As the disease progresses, multiple systems such as respiratory, digestive, and skeletal systems may be affected.

[0003] SMA is an autosomal recessive hereditary disease caused by mutations in the survival motorneuron 1 (SMN1) gene. SMN1 mutations lead to degeneration of alpha motor neurons in the anterior horn cells of the spinal cord, which in turn leads to severe progressive hypotonia and muscle weakness, which can cause early death in patients. The SMN gene has two highly homologous SMN1 and SMN2 genes. It has been found that the SMN1 and SMN2 genes only differ by 5 bases in the region from exon 6 to exon 8 (c.835-44, c.840, INS7+100, INS7+215, c.*239). c.840 is on exon 7, with a homology of more than 99%, and both encode SMN proteins. The SMN1 gene encodes a functional full-length SMN protein, while SMN2 only expresses a small amount of protein with normal function. SMN1 is the pathogenic gene of SMA, and SMN2 is a modified compensatory gene that is associated with the severity of the disease.

[0004] There are two main types of SMA mutation genotypes, 95% are caused by biallelic homozygous deletion of the SMN1 gene; 5% are caused by compound heterozygous mutations of the SMN1 gene, that is, one allele is deleted and the other allele has a minor pathogenic mutation (including point mutation). It is very rare for both SMN1 alleles to be minor pathogenic. Most SMN1 gene deletions are exon 7 and exon 8 deletions, and a small number are only exon 7 deletions. SMN1 gene minor pathogenic variants show different mutation spectrums in patients of different races. At present, about 90 minor pathogenic variants have been reported at home and abroad (http: / / www.hgmd.cf.ac.uk / ), and nearly 30 have been reported in Chinese patients. Among them, there are 7 pathogenic minor variants with a detection frequency ≥2 and confirmed by the American College of Medical Genetics and Genomics (ACMG) pathogenicity assessment. The most common mutations in the Chinese population are c.22dupA (p.Ser8Lys fs*23) in exon 1 and c.683T>A (p.Leu228*) in exon 5, accounting for approximately 31.7% and 17.1% of all point mutations, respectively.

[0005] The carrier frequency of SMA in the population is about 1 / 40-50, and the estimated incidence rate is 1 / 10,000-12,000 live births. It is a common autosomal recessive genetic disease. The high disability and high lethality of SMA have a heavy impact and burden on the patient's family and society. The current listing of disease-modifying treatments and gene therapy drugs has brought great hope to patients. More and more evidence shows that the earlier the presymptomatic gene therapy is performed on the child, the greater the benefit to the patient, and most patients can even show normal motor function development. Early detection through newborn screening is essential to ensure effective treatment before the onset of disease symptoms. Prenatal cases identified through carrier screening will also allow early treatment. Carrier and newborn screening for SMA has been routinely carried out in some countries and regions, and the SMA prevention window has been further advanced.

[0006] The 2020 "Expert Consensus on Genetic Diagnosis of Spinal Muscular Atrophy" (hereinafter referred to as the "Expert Consensus on Genetic Diagnosis") clearly states that biallelic pathogenic variants in SMN1 are the main basis for the diagnosis of SMA, and patients with clinical diagnosis or clinical suspicion of SMA should undergo genetic testing. The target genes for genetic testing are the SMN1 gene and the SMN2 gene. Among them, the detection results of the SMN1 gene copy number and pathogenic variants are used for disease diagnosis or exclusion diagnosis, and the detection results of the SMN2 gene copy number are used as reference indicators for treatment, clinical management and prognosis evaluation after patient diagnosis. Since the SMN1 gene and SMN2 gene are highly homologous in the human genome, genetic testing is required for the SMN1 gene and SMN2 gene respectively. The differential base sites of the full-length transcripts of the SMN1 gene and SMN2 gene in exon 7 (c.840C / T) and exon 8 (c.*239G / A) are used as the main reference sites to distinguish the two loci. Since 95% of SMA patients have homozygous deletion of exon 7 of the SMN1 gene, quantitative analysis of the copy number of exon 7 of the SMN1 gene should be performed first. If there is a homozygous deletion of exon 7 of the SMN1 gene or a homozygous deletion of exons 7 and 8, the patient can be diagnosed as a SMN1 homozygous deletion type patient. For non-homozygous deletion variants, the pathogenic minor variants of the SMN1 gene must also be analyzed and confirmed. For patients with a clear genetic diagnosis, it is necessary for their parents to undergo SMN1 gene testing in order to determine the parents' SMN1 genotype and the source of the patient's mutant gene, and to provide genetic counseling.

[0007] Commonly used methods for detecting the copy number of the SMN1 gene include multiplex ligation probe amplification (MLPA) and quantitative polymerase chain reaction (qPCR). Traditional detection methods for SMA also include bilateral dual allele-specific PCR (AS-PCR), polymerase chain reaction-denaturing high-performance liquid chromatography (PCR-DHPLC), PCR-SSCP, PCR-RFLP, DHPLC, high-resolution melting curve analysis (HRMA), digital PCR, etc. MLPA can clearly distinguish patients, carriers or normal people by directly detecting the copy number of SMN1, and can also detect the copy number of SMN2 at the same time. However, MLPA is complicated to operate, costly, and requires anticoagulated blood to extract purified genomic DNA, which is not suitable for large-scale newborn screening. qPCR uses the housekeeping gene sequence as the internal reference and the Taqman probe method to relatively quantify the copy number of SMN1 exon 7 or 7, 8 to determine whether a deletion mutation has occurred. The operation is simple and the cost is low, but it also requires anticoagulant blood to extract and purify genomic DNA. Direct extraction and detection of dried blood spots will lead to differences in the amplification efficiency of the target gene and the internal reference gene between samples, which may easily lead to false positive and false negative results in carrier detection.

[0008] In the prior art, there are also methods that use the melting curve method of adding fluorescent dyes or fluorescent probes to detect the copy number of the SMN1 gene, but they are all designed by separately designing probes for the target region and the internal reference region, performing melting curve analysis by amplifying the two regions, and detecting the copy number variation of the target region by the ratio of the melting peak intensity of the two regions. The target region and the internal reference region are different gene sequences, and the gene amplification efficiency will be different. In order to reduce the impact of different amplification efficiencies, extremely low dNTP concentrations are used for restriction amplification, but the amount of amplified products will be significantly reduced, affecting the detection sensitivity, and it is difficult to meet the requirements of neonatal dry blood spot trace sample detection. Directly designing fluorescent probes to detect the SMN1 gene and SMN2 gene at the differential base site (c.840C / T) in exon 7 can only detect patients with complete deletion of both copies of SMN1, but cannot detect carriers of single copy deletion, because it is impossible to distinguish between the 1:1 and 2:2 copy numbers of SMN1 and SMN2.

[0009] The above conventional methods mainly use anticoagulated blood samples to detect the copy number of SMN1 exon 7, and cannot detect minor variations in the SMN1 gene. Therefore, there may be missed detections during screening. Studies have shown that high-throughput sequencing technology can simultaneously detect the copy number of the SMN1 gene and screen for minor variations in the SMN gene, but high-throughput sequencing technology is complex to operate, has cumbersome steps, and has high instrument and reagent costs, and is also not suitable for large-scale newborn screening. Summary of the invention

[0010] The purpose of the present invention is to provide a primer probe set and a kit for detecting the SMN1 pathogenic gene of spinal muscular atrophy. The kit provided by the present invention overcomes the shortcomings and defects of the prior art, uses the fluorescent probe melting curve technology to simultaneously detect the SMN1 gene copy number and common point mutations, can detect trace samples including dried blood spots / saliva, can simultaneously detect SMA patients and carriers, and has the advantages of short time consumption, simple operation, strong specificity, high sensitivity, high accuracy and low cost.

[0011] SMN1 gene, the full name is survival motor neuron gene 1.

[0012] The present invention provides a test kit, comprising gene detection liquid A, gene detection liquid B, and gene detection liquid C.

[0013] The kit also includes a pre-treatment solution and an amplification reaction solution.

[0014] The present invention also protects the use of gene detection liquid A, gene detection liquid B and gene detection liquid C in preparing a kit.

[0015] The present invention also protects the use of gene detection solution A, gene detection solution B, gene detection solution C, pre-treatment solution and amplification reaction solution in preparing a kit.

[0016] The gene detection solution A contains primer F4, primer R4, primer F6, primer R6, primer F7, primer R7, probe FP4, probe Q2, probe FP8, probe Q5, probe FP9, probe FP10 and probe Q6.

[0017] Gene detection solution B contains primer F4, primer R4, primer F6, primer R6, primer F5, primer R5, probe FP6, probe Q4, probe FP7, probe FP11, probe Q7, probe FP5 and probe Q3.

[0018] Gene detection solution C contains primer F1, primer R1, primer F2, primer R2, primer F3, primer R3, probe FP1, probe FP2, probe Q1, probe FP3 and probe FB1.

[0019] In gene detection solution A, the molar ratios of primer F4, primer R4, primer F6, primer R6, primer F7, primer R7, probe FP4, probe Q2, probe FP8, probe Q5, probe FP9, probe FP10 and probe Q6 are as follows: 1.5:15:12:1.2:1:10:6:6:6:6:5:5:5.

[0020] In gene detection solution B, the molar ratios of primer F4, primer R4, primer F6, primer R6, primer F5, primer R5, probe FP6, probe Q4, probe FP7, probe FP11, probe Q7, probe FP5 and probe Q3 are as follows: 1.5:15:1:10:1.5:15:7.5:7.5:7.5:5:5:6:6.

[0021] In gene detection solution C, the molar ratios of primer F1, primer R1, primer F2, primer R2, primer F3, primer R3, probe FP1, probe FP2, probe Q1, probe FP3, and probe FB1 are as follows: 1:10:1:10:10:1:5:5:5:5:2.

[0022] The pretreatment solution contains Chelex-100, Triton-X100 and NaOH.

[0023] Specifically, the pretreatment solution contains 5% (mass percentage) Chelex-100, 0.2% (volume percentage) Triton-X100, 40 mM NaOH, and the balance is water.

[0024] The amplification reaction solution contains DNA polymerase, dNTP, dUTP and UDG enzyme.

[0025] Specifically, the composition of the amplification reaction solution for one person (6.0 μl) is shown in Table 4.

[0026] The present invention also protects a primer probe set, which consists of primer F1, primer R1, primer F2, primer R2, primer F3, primer R3, primer F4, primer R4, primer F5, primer R5, primer F6, primer R6, primer F7, primer R7, probe FP1, probe FP2, probe Q1, probe FP3, probe FB1, probe FP4, probe Q2, probe FP5, probe Q3, probe FP6, probe Q4, probe FP7, probe FP8, probe Q5, probe FP9, probe Q6, probe FP10, probe FP11 and probe Q7.

[0027] The present invention also protects the use of the primer probe set in preparing a kit.

[0028] The present invention also protects a kit, comprising the primer probe set.

[0029] The functions of any of the above kits are as follows (a) or (b): (a) detecting SMN1 gene mutation; (b) screening patients with spinal muscular atrophy; (c) screening carriers of spinal muscular atrophy.

[0030] Primer F1 is the single-stranded DNA molecule shown in SEQ ID NO: 1; primer R1 is the single-stranded DNA molecule shown in SEQ ID NO: 2; primer F2 is the single-stranded DNA molecule shown in SEQ ID NO: 3; primer R2 is the single-stranded DNA molecule shown in SEQ ID NO: 4; primer F3 is the single-stranded DNA molecule shown in SEQ ID NO: 5; primer R3 is the single-stranded DNA molecule shown in SEQ ID NO: 6; primer F4 is the single-stranded DNA molecule shown in SEQ ID NO: 7; primer R4 is the single-stranded DNA molecule shown in SEQ ID NO: 8; primer F5 is the single-stranded DNA molecule shown in SEQ ID NO: 9; primer R5 is the single-stranded DNA molecule shown in SEQ ID NO: 10; primer F6 is the single-stranded DNA molecule shown in SEQ ID NO: 11; primer R6 is the single-stranded DNA molecule shown in SEQ ID NO: 12; primer F7 is the single-stranded DNA molecule shown in SEQ ID NO: 13; primer R7 is the single-stranded DNA molecule shown in SEQ ID NO: 14.

[0031] Probe FP1 is a single-stranded DNA molecule shown in SEQ ID NO: 15; probe FP2 is a single-stranded DNA molecule shown in SEQ ID NO: 16; probe Q1 is a single-stranded DNA molecule shown in SEQ ID NO: 17; probe FP3 is a single-stranded DNA molecule shown in SEQ ID NO: 18; probe FB1 is a single-stranded DNA molecule shown in SEQ ID NO: 19; probe FP4 is a single-stranded DNA molecule shown in SEQ ID NO: 20; probe Q2 is a single-stranded DNA molecule shown in SEQ ID NO: 21; probe FP5 is a single-stranded DNA molecule shown in SEQ ID NO: 22; probe Q3 is a single-stranded DNA molecule shown in SEQ ID NO: 23; probe FP6 is a single-stranded DNA molecule shown in SEQ ID NO: 24; probe Q4 is a single-stranded DNA molecule shown in SEQ ID NO: 25; probe FP7 is a single-stranded DNA molecule shown in SEQ ID NO: 26; probe FP8 is a single-stranded DNA molecule shown in SEQ ID NO: 27; probe Q5 is a single-stranded DNA molecule shown in SEQ ID NO: 28; probe FP9 is a single-stranded DNA molecule shown in SEQ ID NO: 29; probe Q6 is a single-stranded DNA molecule shown in SEQ ID NO: 30 The probe FP10 is the single-stranded DNA molecule shown in SEQ ID NO: 31; the probe FP11 is the single-stranded DNA molecule shown in SEQ ID NO: 32; and the probe Q7 is the single-stranded DNA molecule shown in SEQ ID NO: 33.

[0032] Probe FP4, probe FP8, probe FP9 and probe FP10 are respectively labeled with different fluorescent groups (ie, the four probes have four fluorescent groups).

[0033] Probe FP6, probe FP7, probe FP11 and probe FP5 are respectively labeled with different fluorescent groups (ie, the four probes have four fluorescent groups).

[0034] Probe FP1, probe FP2 and probe FP3 are respectively labeled with different fluorescent groups (ie, the four probes have three fluorescent groups).

[0035] Probe FP1, 5' end labeled with a fluorescent group, 3' end labeled with a fluorescent quencher group;

[0036] Probe FP2, 5' end labeled with a fluorescent group, 3' end modified with C3 Spacer;

[0037] Probe Q1, 3' end labeled with a fluorescent quencher group;

[0038] Probe FP3, 5' end labeled with a fluorescent group, 3' end labeled with a fluorescent quencher group;

[0039] Probe FP4, 5' end labeled with a fluorescent group, 3' end modified with C3 Spacer;

[0040] Probe Q2, 3' end labeled with a fluorescent quencher group;

[0041] Probe FP5, 5' end labeled with a fluorescent group, 3' end modified with C3 Spacer;

[0042] Probe Q3, 3' end labeled with a fluorescent quencher group;

[0043] Probe FP6, 3′ end labeled with a fluorescent group;

[0044] Probe Q4, 5' end labeled with a fluorescent quencher group, 3' end labeled with a fluorescent quencher group;

[0045] Probe FP7, 5' end labeled with a fluorescent group, 3' end modified with C3 Spacer;

[0046] Probe FP8, 5' end labeled with a fluorescent group, 3' end modified with C3 Spacer;

[0047] Probe Q5, 3' end labeled with a fluorescent quencher group;

[0048] Probe FP9, 3′ end labeled with a fluorescent group;

[0049] Probe Q6, 5' end labeled with a fluorescent quencher group, 3' end labeled with a fluorescent quencher group;

[0050] Probe FP10, 5' end labeled with a fluorescent group, 3' end modified with C3 Spacer;

[0051] Probe FP11, 5' end labeled with a fluorescent group, 3' end modified with C3 Spacer;

[0052] The 3' end of probe Q7 is labeled with a fluorescence quencher group.

[0053] Probe FP1, 5′ end labeled with FAM and 3′ end labeled with BHQ2;

[0054] Probe FP2, 5' end labeled with CY5, 3' end modified with C3 Spacer;

[0055] Probe Q1, 3′ end labeled with BHQ2;

[0056] Probe FP3, 5′ end labeled with ROX and 3′ end labeled with BHQ2;

[0057] Probe FB1, the 12th and 13th nucleotides were modified with locked nucleic acid, and the 3' end was modified with C3 Spacer;

[0058] Probe FP4, 5' end labeled with CY5, 3' end modified with C3 Spacer;

[0059] Probe Q2, 3′ end labeled with BHQ2;

[0060] Probe FP5, 5′ end labeled with ROX, 3′ end modified with C3 Spacer;

[0061] Probe Q3, 3′ end labeled with BHQ2;

[0062] Probe FP6, 3′ end labeled with FAM;

[0063] Probe Q4, 5′ end labeled with BHQ1 and 3′ end labeled with BHQ1;

[0064] Probe FP7, 5' end labeled with HEX, 3' end modified with C3 Spacer;

[0065] Probe FP8, 5′ end labeled with ROX, 3′ end modified with C3 Spacer;

[0066] Probe Q5, 3′ end labeled with MGB;

[0067] Probe FP9, 3′ end labeled with FAM;

[0068] Probe Q6, 5′ end labeled with BHQ1 and 3′ end labeled with BHQ1;

[0069] Probe FP10, 5' end labeled with HEX, 3' end modified with C3 Spacer;

[0070] Probe FP11, 5' end labeled with CY5, 3' end modified with C3 Spacer;

[0071] Probe Q7, 3' end labeled with MGB.

[0072] The working principles of the kit provided by the present invention are principle one, principle two and principle three.

[0073] The schematic diagrams of Principle 1 and Principle 2 are shown in Figure 1 .

[0074] Principle 1 is as follows:

[0075] For the two target sites of SMN1 gene exon 7 (c.840C / T; SMN1 gene is c.840C, SMN2 gene is c.840T) and exon 8 (c.*239G / A; SMN1 gene is c.*239G, SMN2 gene is c.*239A), upstream and downstream primers were designed, and asymmetric PCR was used to amplify single-stranded DNA containing the target region.

[0076] The fluorescent detection probe is designed according to the target, which is a self-quenching probe or an adjacent quenching probe group. One end of the self-quenching probe is labeled with a fluorescent group, and the other end is labeled with a fluorescent quenching group; when there is no target, the probe curls naturally, the fluorescent group and the fluorescent quenching group are close, so that the fluorescence is quenched; when there is a target, the probe hybridizes with the target, the fluorescent group and the fluorescent quenching group are separated, and a fluorescent signal appears. The adjacent quenching probe group consists of two probes, one is a fluorescent probe (labeled with a fluorescent group, and the target area of ​​the fluorescent probe covers the target site), and the other is a quenching probe (labeled with a fluorescent quenching group, and the target area of ​​the fluorescent quenching probe is adjacent to the target area of ​​the fluorescent probe); when the target is present, the fluorescent probe and the quenching probe hybridize on the target, the distance is close, and the fluorescent group is quenched by the fluorescent quenching group; when the target is not present, the fluorescent probe and the quenching probe are separated, and a fluorescent signal appears on the fluorescent probe.

[0077] The different typing of the c.840 site and the c.*239 site and the different probe binding forces lead to differences in Tm in the melting curve, so the genotyping can be determined based on the Tm value distribution. Using the differential sites of the SMN1 gene and the SMN2 gene, the probe completely matches the SMN1 gene and does not completely match the SMN2 gene. Therefore, the amplified product of the SMN1 gene produces a melting peak with a high Tm value, and the amplified product of the SMN2 gene produces a melting peak with a low Tm value. If the high Tm value melting peak is missing, it means that both copies of the SMN1 gene are completely missing. If the high Tm value melting peak and the low Tm value melting peak have equal intensities, it may be "SMN1 gene: SMN2 gene copy number is 1:1" or "SMN1 gene: SMN2 gene copy number is 2:2", so it cannot be directly determined whether it is a single copy of the SMN1 gene deletion.

[0078] Principle 2 is as follows:

[0079] In order to analyze the single copy deletion (carrier) of SMN1 gene, the homologous gene amplification comparative analysis method was used. The target region (SMN1-Target) was selected near the exon 7 region of SMN1 gene, and the reference region (SMN1-Ref) with high homology to the target region was compared in the whole genome. The number of highly homologous regions was no more than 2. PCR amplification upstream and downstream primers were designed for the screened highly homologous regions, and the same primers were used to simultaneously amplify SMN1-Target and SMN1-Ref. Because the SMN1 gene and the SMN2 gene are highly homologous, in order to avoid interference from the SMN2 gene, blocking primers were designed based on the differential site INS7+100 between the SMN1 gene and the SMN2 gene (INS7+100A for SMN1 and INS7+100G for SMN2) (the 3' end of the primer differs from the SMN1 gene by 1 base and from the SMN2 gene by 2 bases), and a blocking probe was designed (the blocking probe covers the INS7+100 site and the 3' end of the primer, the blocking probe completely matches the SMN2 gene INS7+100G, and the INS7+100G base is modified with locked nucleotides to ensure that the blocking probe preferentially binds to the SMN2 gene to avoid interference from the SMN2 gene). A fluorescent detection probe is designed, and the hybridization region of the fluorescent detection probe selects a sequence with base differences between SMN1-Target and SMN1-Ref, so that SMN1-Target and SMN1-Ref are distinguished by different Tm melting peaks. The melting peak with a high Tm value is the amplification product of the SMN1 gene, and the melting peak with a low Tm value is the amplification product of the homologous reference region of the genome. When the SMN1 gene has a copy number loss, the template ratio of the SMN1 gene to the homologous reference region changes from 2:2 to 1:2 or 0:2. Because the same amplification primers are used, when the template ratio changes significantly, the ratio of the amplified products also changes significantly, so that the melting peak intensity ratio changes, thereby detecting single-copy loss and double-copy loss of the SMN1 gene.

[0080] Principle three is as follows:

[0081] In order to simultaneously analyze the common mutation sites of the SMN1 gene, amplification primers were designed for the most common mutation sites in the Chinese population (see Table 1). Primers were designed to amplify the corresponding mutation sites. Fluorescence detection probes were designed according to the target, which were self-quenching probes or adjacent quenching probe sets. If a mutation occurs at the detection site, the probe melting peak will change, with a low Tm value melting peak or a high Tm value melting peak, thereby determining whether it is wild type, heterozygous mutant type or homozygous mutant type.

[0082] Table 1

[0083] Serial number Mutation site and mutation form 1 c.22dupA(p.Ser8Lys fs*23) 2 c.683T>A(p.Leu228*) 3 c.689C>T(p.Ser230Leu) 4 c.863G>T(p.Gly279Glufs*5) 5 c.400G>A(p.Glu134Lys) 6 c.835-5T>G(p.Gly279Glufs*5) 7 c.463_464delAA(p.Asn155Cysfs*5) 8 c.5C>G (p.Ala2Gly) 9 c.40G>T(p.Glu13X) 10 c.43C>T(p.Glu14X) 11 c.56delT(p.Val19Glyfs*21)

[0084] The kit provided by the present invention can detect double copy deletion, single copy carrier and common mutations (i.e. mutations shown in Table 1) of the SMN1 gene, thereby reducing the missed detection rate. The kit provided by the present invention adopts homologous region amplification, uses normal dNTP concentration, and specifically detects the copy number variation of the target region without affecting the detection sensitivity. The gene detection reagents in the prior art require more blood samples and purify genomic DNA, which are not suitable for neonatal dry blood spot screening. The kit provided by the present invention can directly pre-treat trace blood samples (3μl blood or 3mm dry blood spots), and the treatment liquid directly performs subsequent amplification detection, without the need for cumbersome steps such as genomic DNA purification, saving cost and time, and is suitable for neonatal dry blood spot screening detection. The present invention provides a kit that can be used for SMN1 gene carrier detection for trace saliva samples, which is suitable for carrier screening in normal people and pregnant people, and effectively carries out three-level prevention and control of SMA.

[0085] The beneficial effects of the present invention are as follows: (1) SMN1 gene detection is completed through three PCR reactions, and homozygous deletions of exons 7 and 8, single copy deletions of exon 7 and common mutations (i.e., mutations shown in Table 1) are detected simultaneously, which can effectively reduce missed detections, and the detection cost is low, the throughput is high, the operation is convenient, and the time is short (detection is completed in 2.5 hours); (2) The sensitivity reaches 2ng; (3) Direct detection of trace dried blood spots can be realized, which is beneficial for newborn screening; (4) Direct detection of trace saliva samples can be realized, which is beneficial for large-scale carrier screening in a non-invasive manner; (5) Amplification primers and probes of the region near SMN1 exon 7 are designed according to the homologous sequence of the genome by using fluorescent probe multiple melting curves and homologous gene comparison analysis to realize CNV detection, which improves the detection specificity relative to the Taqman probe method. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 The principle of amplification of exon 7 and exon 8 regions of the SMN1 gene.

[0087] Figure 2 To inhibit the proliferation of SMN2.

[0088] Figure 3 Comparison of probe systems for detecting point mutations in the SMN1 gene.

[0089] Figure 4 Comparison of probe systems for detecting exon 7 of the SMN1 gene.

[0090] Figure 5 Comparison of probe systems for detecting exon 8 of the SMN1 gene.

[0091] Figure 6 Comparison of probe systems for SMN1 gene copy number detection.

[0092] Figure 7 Plasmid for detecting point mutations in the SMN1 gene.

[0093] Figure 8 Plasmid for detecting point mutations in the SMN1 gene.

[0094] Fig. 9 This is a diagram showing the clinical testing results for SMA patients.

[0095] Fig.10 This is a diagram showing the results of clinical SMA carrier detection.

[0096] Fig.11 This is a diagram showing the detection effect of normal clinical samples.

[0097] Fig.12 Detection of different SMN1 copy numbers in clinical samples.

[0098] Fig.13 Detection of heterozygous mutation of SMN1 gene c.22dupA.

[0099] Fig.14 Detection of heterozygous mutation c.400G>A in SMN1 gene.

[0100] Fig.15 Detection of SMN1 gene c.863G>T heterozygous mutation.

[0101] Fig.16 This is the effect of different SMN2 copy numbers on SMN1 detection. DETAILED DESCRIPTION

[0102] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Unless otherwise specified, the quantitative tests in the following examples are all set to three repeated experiments, and the results are averaged.

[0103] Example 1. Preparation and use of SMN1 gene detection kit

[0104] 1. Primer design, screening and preparation

[0105] Primers were designed for the mutation site regions in exon 7 and exon 8 of the SMN1 and SMN2 genes, and the primers with the best performance (specificity, sensitivity) were obtained through combination and screening in preliminary experiments. The primers designed according to principle 1 are as follows: upstream primer F1 and downstream primer R1 are used to amplify the exon 7 region (including the c.840 site) of the SMN1 and SMN2 genes; upstream primer F2 and downstream primer R2 are used to amplify the exon 8 region (including the c.*239 site) of the SMN1 and SMN2 genes.

[0106] The sequence from exon 7 to IVS7+100 of the SMN1 gene was selected, and a homologous region was searched in other regions of the whole genome, and a homologous region was found. The sequence between IVS7+21 and IVS7+126 of the SMN1 gene was selected as the detection target sequence SMN1-Target, and the chromosome position was Chr5:70951950-70952192; the homologous sequence on the genome was located at Chr9:20332239-20332475, and was selected as the reference sequence SMN1-Ref. According to the homologous sequence alignment information, the consistent region was selected in the upstream primer F3 as the upstream primer region for amplification, and cgc was added at the 5' end to increase the primer Tm value. The blocking primer principle was used in the downstream primer R3 to mutate a base T near the 3' end into c and cgg was added at the 5' end to increase the primer Tm value. The primers designed according to principle 2 are as follows: upstream primer F3 and downstream primer R3, which are used to simultaneously amplify the SMN1 target sequence region and the homologous reference sequence region on chromosome 9.

[0107] Primers were designed based on sequence analysis, and the effects of primers were compared through preliminary experiments to screen and obtain the optimal primers. The primers designed according to principle three are as follows: upstream primer F4 and downstream primer R4 are used to amplify the region including 5 variant sites (c.22dupA, c.5C>G, c.40G>T, c.43C>T, c.56delT) in the SMN1 gene; upstream primer F5 and downstream primer R5 are used to amplify the region including 2 variant sites (c.683T>A, c.689C>T) in the SMN1 gene; upstream primer F6 and downstream primer R6 are used to amplify the region including 2 variant sites (c.863G>T, c.835-5T>G) in the SMN1 gene; upstream primer F7 and downstream primer R7 are used to amplify the region including 2 variant sites (c.463_464delAA, c.400G>A) in the SMN1 gene.

[0108] The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer powder was dissolved in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) and quantified to 20 μM using a micro-spectrophotometer.

[0109] The relevant information of primers and nucleotide sequences are shown in Table 2.

[0110] Table 2 Amplification sites or regions and their corresponding primers

[0111]

[0112] 2. Preparation of probe

[0113] The fluorescence detection probe is a self-quenching probe or an adjacent quenching probe group.

[0114] The self-quenching probe is used alone, and the probe is modified with a fluorescent group and a fluorescent quenching group at the 5' end and the 3' end respectively. The fluorescent group is any one of FAM, HEX, CY5, and ROX, and the fluorescent quenching group is any one of BHQ1, BHQ2, and BHQ3 that can be used in conjunction with the fluorescent group.

[0115] The adjacent quenching probe group is used in combination with a fluorescent probe and a quenching probe. The 5' end of the fluorescent probe is labeled with a fluorescent group and the 3' end is modified with C3 Spacer, and the 3' end of the quenching probe matched with it is labeled with a fluorescent quenching group. Alternatively, the 3' end of the fluorescent probe is labeled with a fluorescent group, and the 5' end of the quenching probe matched with it is labeled with a fluorescent quenching group and the 3' end is modified with C3 Spacer (for a quenching probe of a certain fluorescent probe) or the 3' end is labeled with a fluorescent quenching group (for a quenching probe of two fluorescent probes). The fluorescent group is any one of FAM, HEX, CY5, and ROX, and the fluorescent quenching group is any one of BHQ1, BHQ2, BHQ3 or MGB that can be used in combination with the fluorescent group. C3 Spacer is a blocking group to prevent the probe from being extended as a primer.

[0116] The blocking probe was not modified with a fluorescent group, but the 3' end was modified with a C3 Spacer.

[0117] Probe FP1 is a self-quenching probe that covers the c.840 site of the SMN1 gene. The sequence completely matches the SMN1 gene, but does not match the SMN2 gene at the c.840 site. Therefore, the Tm value of the binding of probe FP1 to the SMN2 amplification sequence is lower than the Tm value of the binding of probe FP1 to the SMN1 amplification sequence, thereby detecting exon 7 of the SMN1 gene and SMN2 gene.

[0118] Probe FP2 is a fluorescent probe, and probe Q1 is a quenching probe. The two constitute an adjacent quenching probe group, covering the c.*239 site of the SMN1 gene. The sequence completely matches the SMN1 gene, but does not match the SMN2 gene at the c.*239 site. Therefore, the Tm value of the binding of probe FP2 to the SMN2 amplification sequence is lower than the Tm value of the binding of probe FP2 to the SMN1 amplification sequence, thereby detecting exon 8 of the SMN1 gene and SMN2 gene.

[0119] Probe FP3 is a self-quenching probe, and its sequence is completely complementary to the target sequence in SMN1-Target. It differs by 4 bases from the homologous reference sequence in SMN1-Ref. Therefore, the amplification product corresponding to the SMN1 target sequence SMN1-Target of probe FP3 produces a high Tm value melting peak, while the amplification product corresponding to the homologous reference sequence SMN1-Ref forms a low Tm melting peak. The copy number ratio of the SMN1 gene and the homologous reference sequence is detected based on the ratio of the two melting peaks, thereby realizing the detection of single-copy deletion and double-copy deletion of the SMN1 gene.

[0120] Probe FB1 is a blocking probe designed for the IVS7+100 site of the SMN1 / 2 gene. It completely matches the IVS7+100 site of the SMN2 gene. It is modified with a locked nucleic acid at the differential site IVS7+100, preferentially binds to the SMN2 gene, increases the Tm value difference, and improves the blocking effect. The 3' end is modified with C3 Spacer.

[0121] Probe FP4 is a fluorescent probe and probe Q2 is a quenching probe. The two constitute an adjacent quenching probe group, covering the c.22dupA site of the SMN1 gene. The sequence completely matches the wild type of the SMN1 gene. If a mutation occurs at the detection site, the probe melting peak will change, and a melting peak with a low Tm value will appear, thereby determining whether it is a wild type, a heterozygous mutant type, or a homozygous mutant type.

[0122] Probe FP5 is a fluorescent probe, and probe Q3 is a quenching probe. The two constitute an adjacent quenching probe group, covering the c.683T>A site and c.689C>T site of the SMN1 gene. The sequence completely matches the c.683T>A wild type and c.689C>T mutant type of the SMN1 gene, that is, the probe sequence is the c.683T and c.689T genotypes; if the detection site c.683T>A mutates, the probe melting peak will change, and a melting peak with a low Tm value will appear; if the detection site c.689C>T mutates, the probe melting peak will change, and a melting peak with a high Tm value will appear; thereby determining whether it is wild type, heterozygous mutant type or homozygous mutant type.

[0123] Probe FP6 is a fluorescent probe, and probe Q4 is a quenching probe (probe Q4 is also compatible with probe FP6 and probe FP7). The two constitute an adjacent quenching probe group, covering the c.5C>G site of the SMN1 gene. The sequence completely matches the wild type of the SMN1 gene. If a mutation occurs at the detection site, the probe melting peak will change, and a melting peak with a low Tm value will appear, thereby determining whether it is a wild type, a heterozygous mutant type, or a homozygous mutant type.

[0124] Probe FP7 is a fluorescent probe, and probe Q4 is a quenching probe (probe Q4 is also matched with probe FP6 and probe FP7). The two constitute an adjacent quenching probe group, covering the SMN1 gene c.40G>T site, c.43C>T site and c.56delT site. The sequence is completely matched with the SMN1 gene c.40G>T site mutant type, c.43C>T site wild type and c.56delT site wild type, that is, the probe sequence is c.40T, c.43C and c.56T genotypes; if the detection site c.40G>T mutates, the probe melting peak will change, and a melting peak with a high Tm value will appear; if the detection site c.43C>T mutates, the probe melting peak will change, and a melting peak with a low Tm value will appear; if the detection site c.56delT mutates, the probe melting peak will change, and a melting peak with a low Tm value will appear; thereby determining whether it is a wild type, a heterozygous mutant type or a homozygous mutant type.

[0125] Probe FP8 is a fluorescent probe and probe Q5 is a quenching probe. The two constitute an adjacent quenching probe group, covering the c.863G>T site of the SMN1 gene. The sequence completely matches the wild type of the SMN1 gene. If a mutation occurs at the detection site, the probe melting peak will change, and a melting peak with a low Tm value will appear, thereby determining whether it is wild type, heterozygous mutant type, or homozygous mutant type.

[0126] Probe FP9 is a fluorescent probe, and probe Q6 is a quenching probe (probe Q6 is also compatible with probe FP9 and probe FP10). The two constitute an adjacent quenching probe group, covering the c.400G>A site of the SMN1 gene. The sequence completely matches the wild type of the SMN1 gene. If a mutation occurs at the detection site, the probe melting peak will change, and a melting peak with a low Tm value will appear, thereby determining whether it is a wild type, a heterozygous mutant type, or a homozygous mutant type.

[0127] Probe FP10 is a fluorescent probe, and probe Q6 is a quenching probe (probe Q6 is also matched with probe FP9 and probe FP10). The two constitute an adjacent quenching probe group, covering the c.463_464delAA site of the SMN1 gene. The sequence completely matches the mutant type of the SMN1 gene. If a mutation occurs in the detection site, the probe melting peak will change, and a melting peak with a high Tm value will appear, thereby determining whether it is wild type, heterozygous mutant type or homozygous mutant type.

[0128] Probe FP11 is a fluorescent probe and probe Q7 is a quenching probe. The two constitute an adjacent quenching probe group, covering the c.835-5T>G site of the SMN1 gene. The sequence completely matches the wild type of the SMN1 gene. If a mutation occurs at the detection site, the probe melting peak will change and a melting peak with a low Tm value will appear, thereby determining whether it is wild type, heterozygous mutant type or homozygous mutant type.

[0129] The probe was synthesized and modified by Sangon Biotech (Shanghai) Co., Ltd. The probe powder was dissolved in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) and quantified to 20 μM using a micro-spectrophotometer.

[0130] The relevant information and nucleotide sequences of the probes are shown in Table 3.

[0131] Table 3 Probe related information and nucleotide sequence

[0132]

[0133] 3. Preparation of SMN1 gene detection kit

[0134] The SMN1 gene detection kit (100 servings) consists of the following components: 15 ml pretreatment solution, 2 ml amplification reaction solution, 650 μl gene detection solution A, 650 μl gene detection solution B, 650 μl gene detection solution C, standard control, and 500 μl negative control solution.

[0135] Pretreatment solution: contains 5% (mass percentage) Chelex-100, 0.2% (volume percentage) Triton-X100, 40mM NaOH, and the balance is water. 100Chelating Resin): Bio-rad, Catalog No. 1421253. Triton-X100: Shanghai Biotech Co., Ltd., A110694-0100.

[0136] The composition of the amplification reaction solution for one person (6.0 μl) is shown in Table 4.

[0137] Table 4

[0138] Reagents 1 serving (μl) 5X Hotstart Taq Buffer 5 Hotstart Taq DNA Polymerase 0.4 dNTP Mixture 0.5 dUTP 0.05 2U / μl UDG enzyme 0.05 total 6.0

[0139] The reagents in Table 4 are all products of Shanghai Bioengineering Co., Ltd. Hotstart Taq DNA Polymerase (specification: 5U / μl) and 5X Hotstart Taq Buffer are supporting products, with the catalog number B110003-2500. dNTP Mixture (specification: 10mM), catalog number B110044-0005. dUTP (specification: 100mM), catalog number B110052-0250. UDG enzyme (specification: 2U / μl), catalog number B110042-0200.

[0140] The composition of gene detection solution A for one reaction (total volume of 6 μl) is shown in Table 5. The primers and probes in gene detection solution A are used to detect c.22dupA, c.863G>T, c.400G>A, and c.463_464delAA.

[0141] Table 5

[0142]

[0143] The composition of gene detection solution B for one reaction (total volume of 6 μl) is shown in Table 6. The primers and probes in gene detection solution B were used to detect c.5C>G, c.40G>T, c.43C>T, c.56delT, c.835-5T>G, c.683T>A, and c.689C>T.

[0144] Table 6

[0145]

[0146]

[0147] The composition of gene detection solution C for one reaction (total volume of 6 μl) is shown in Table 7. The primers and probes in gene detection solution C are used to detect exon 7 and exon 8 of the SMN1 gene, as well as SMN1-Target and SMN1-Ref.

[0148] Table 7

[0149]

[0150] Standard control: normal human dried blood spots (SMN1 gene and SMN2 gene are both wild type).

[0151] Negative control solution: deionized water.

[0152] IV. How to use the kit

[0153] 1. Pre-treatment

[0154] Take the sample to be tested (the sample to be tested is: 3μl whole blood, 10μl saliva or 3mm dried blood spot), add 100μl pretreatment solution, incubate at 95℃ for 10min, then centrifuge at 2500rpm for 5min, and collect the supernatant, which is the sample template solution.

[0155] Take the standard control, add 100 μl of pretreatment solution, incubate at 95°C for 10 min, then centrifuge at 2500 rpm for 5 min, and collect the supernatant, which is the standard control solution.

[0156] 2. Preparation of detection reaction system

[0157] Mix 6.0 μl of amplification reaction solution, 2 μl of sample template solution and 6 μl of gene detection solution A, add deionized water to 25 μl, and centrifuge to remove bubbles to obtain sample detection reaction system A.

[0158] Mix 6.0 μl of amplification reaction solution, 2 μl of sample template solution and 6 μl of gene detection solution B, add deionized water to 25 μl, and centrifuge to remove bubbles to obtain sample detection reaction system B.

[0159] Mix 6.0 μl of amplification reaction solution, 2 μl of sample template solution and 6 μl of gene detection solution C, add deionized water to 25 μl, and centrifuge to remove bubbles to obtain sample detection reaction system C.

[0160] The standard control solution replaced the sample template solution to obtain three standard control detection reaction systems.

[0161] The negative control solution replaced the sample template solution to obtain three standard control detection reaction systems.

[0162] 3. Detection

[0163] Fluorescence quantitative PCR instrument (Shanghai Hongshi SLAN-96S) was used to perform PCR amplification and melting curve analysis on the above detection reaction system.

[0164] PCR amplification program: 50°C for 2 min, 95°C for 10 min; 95°C for 20 s, 56°C for 60 s, 45 cycles.

[0165] Melting curve analysis program: 95°C for 1 min, 37°C for 3 min; heating from 40°C to 85°C at a rate of 0.04°C / s, performing melting curve analysis, and collecting the fluorescence signals of each fluorescent group at the same time.

[0166] 4. Results Analysis

[0167] The Tm value of each channel of the reaction detection system is obtained by the configuration software of the fluorescence quantitative PCR instrument according to the fluorescence signal. The Tm value is based on the value automatically determined by the instrument. However, when the instrument cannot give a Tm value, the Tm value can be obtained by adjusting the baseline or manually determining it.

[0168] Quality control: For standard controls, each corresponding fluorescent channel in the reaction system must have a melting peak with Tm within the reference value range (see Table 8 for the reference value range of the melting peak Tm of the standard control); for negative control solutions, each corresponding fluorescent channel in the reaction system must have no clear melting peak. If the quality control is met, the detection amplification system is valid.

[0169] For the sample to be tested, melting peaks appear in each fluorescent signal channel in the reaction system. The melting peaks are compared with the corresponding melting peaks of the standard control, and the SNP typing is determined based on the Tm difference. If there is only a wild-type peak, the genotype of the corresponding gene locus is a homozygous wild type. If there is only a mutant peak, the genotype of the corresponding gene locus is a homozygous mutant type. If both wild-type peaks and mutant peaks appear, the corresponding gene locus is a heterozygous type of wild type and mutant type.

[0170] Table 8 Reference value range of standard control melting peak Tm

[0171]

[0172] Reaction system A and reaction system B were used to detect the common mutation sites of the SMN1 gene listed in Table 1. The analysis scheme was as follows: the Tm value of each channel of the sample detection reaction system was recorded as Tm, and the Tm value of each channel of the standard control detection reaction system was recorded as Tm 标准对照 , through the formula ΔTm=Tm-Tm 标准对照 Obtain the ΔTm value, and determine whether the sample to be tested has the corresponding gene mutation, and the type of mutation according to the ΔTm value. According to the changes in the Tm values ​​of the melting peaks of the sample to be tested and the standard control in each channel in the multicolor probe melting curve analysis results, it is judged whether the sample to be tested contains the corresponding gene mutation, and the type of gene mutation. It involves reading the Tm values ​​of the sample to be tested and the standard control (automatically obtained by the configuration software of the fluorescent PCR instrument), and the standard control is used as a correction to reduce the melting point error caused by different instruments and personnel operations. Subtract the Tm value of each channel of the standard control from the Tm value of each channel of the sample to be tested to obtain the ΔTm of each channel, and judge whether the sample to be tested contains mutations and the type of mutations according to the ΔTm value of each channel (Table 9 and Table 10).

[0173] Table 9

[0174]

[0175] Table 10

[0176]

[0177]

[0178] Reaction system C was used to detect the copy number variation of exon 7 and exon 8 of the SMN1 gene.

[0179] FAM fluorescence channel: the high Tm melting peak is SMN1-Exon7, and the low Tm melting peak is SMN2-Exon7; when exon 7 of both SMN1 gene and SMN2 gene are present in normal samples, both the high Tm melting peak and the low Tm melting peak in the FAM channel have signals; if the high Tm melting peak is missing, it indicates a double copy loss of SMN1-Exon7; if the low Tm melting peak is missing, it indicates a double copy loss of SMN2-Exon7.

[0180] CY5 fluorescence channel: the high Tm melting peak is SMN1-Exon8, and the low Tm melting peak is SMN2-Exon8; when exon 8 of both SMN1 gene and SMN2 gene are present in normal samples, both the high Tm melting peak and the low Tm melting peak of the CY5 channel have signals; the absence of the high Tm melting peak indicates a double copy loss of SMN1-Exon8; the absence of the low Tm melting peak indicates a double copy loss of SMN2-Exon8.

[0181] ROX fluorescence channel:

[0182] The high Tm melting peak is SMN1-Target, and the low Tm melting peak is SMN1-Ref;

[0183] When SMN1 is not deleted in normal samples, both the high Tm melting peak and the low Tm melting peak of the ROX channel have signals;

[0184] When both copies of SMN1 are missing, the high Tm melting peak of the ROX channel is missing;

[0185] When a single copy of SMN1 is missing (carrier), the intensity ratio of the high Tm melting peak and the low Tm melting peak changes, Rm (SMN1-Target) is the intensity of the high Tm melting peak, Rm (SMN1-Ref) is the intensity of the low Tm melting peak, ROX Ratio = Rm (SMN1-Target) / Rm (SMN1-Ref); when 0.75≤ROX Ratio≤1.05, the copy number of the SMN1 exon 7 region is 1 copy; when 1.45≥ROX Ratio≥1.2, the copy number of the SMN1 exon 7 region is 2; when the ROX Ratio is between 1.05-1.2, the copy number analysis result is in the gray zone, and the sample needs to be retested.

[0186] Example 2, Primer Optimization Process (provided as an example, the effect is reflected in the anti-SMN2 gene interference effect)

[0187] Because the SMN1 gene and the SMN2 gene are highly homologous, in order to avoid interference from the SMN2 gene, blocking primers were designed based on the differential site INS7+100 between the SMN1 gene and the SMN2 gene (INS7+100A for SMN1 and INS7+100G for SMN2). The 3' end of the primer differs from the SMN1 gene by 1 base and from the SMN2 gene by 2 bases. A blocking probe was also designed, covering the INS7+100 site and the 3' end of the primer. The blocking probe completely matched the INS7+100G of the SMN2 gene, and the INS7+100G base was modified with a locked nucleotide to ensure that the blocking probe preferentially binds to the SMN2 gene to avoid interference from the SMN2 gene.

[0188] Downstream primers were designed for the differential site INS7+100, which were reverse complementary to the original sequence. The third-to-last base of the reverse primer R3 at the 3' end was changed from the wild-type T to C. The purpose was to increase the amplification difference between the SMN1 gene and the SMN2 gene. There was one base difference with the SMN1 gene and two base differences with the SMN2 gene at the 3' end, which were the third-to-last base C and the last base T. Therefore, the amplification efficiency of the SMN2 gene can be greatly reduced to prevent interference from the SMN2 gene.

[0189] The control primers are reverse primer R3b and reverse primer R3c. Reverse primer R3b differs from SMN1 and SMN2 genes only at the last base T at site INS7+100. Reverse primer R3c has one less base T at the 3' end, and can bind to both SMN1 and SMN2 genes for amplification.

[0190] R3: 5'-cggTGTTTTTACATTAACCTTTCAACT c TT-3'.

[0191] R3b: 5'-cggTGTTTTTACATTAACCTTTCAACTTTT-3'.

[0192] R3c: 5'-cggTGTTTTTACATTAACCTTTCAACTTT-3'.

[0193] In order to compare the anti-interference effects of the designed blocking primers and blocking probes on the SMN2 gene, 6 experimental groups were set up for testing.

[0194] The probe FB1 targeting the SMN2 gene was added to the experimental groups 1-3 (see Table 3 for probe FB1), and the probe FB1 was not added to the experimental groups 4-6. The formula of the amplification reaction solution and the pretreatment solution is shown in step 3 of Example 1. The preparation method of the gene detection solution is shown in Table 11.

[0195] Preparation method of sample template solution: 3μl of whole blood of the subject, add 100μl of pretreatment solution, incubate at 95℃ for 10min, then centrifuge at 2500rpm for 5min, collect the supernatant, which is the sample template solution. The subjects are confirmed SMN1 double copy deletion subjects (patients), SMN1 single copy deletion subjects (carriers), and SMN1 normal subjects, representing 0 copy, 1 copy, and 2 copy samples of SMN1 gene exon 7, respectively. Mix 6.0μl amplification reaction solution, 2μl sample template solution and gene detection solution, add deionized water to 25μl, centrifuge to remove bubbles, and the sample detection reaction system is obtained.

[0196] Perform detection according to step 3 of step 4 of Example 1.

[0197] Table 11 Composition of Gene Detection Solution

[0198]

[0199] For samples (patients) with 0 copies of exon 7 of the SMN1 gene, since only SMN2 exon 7 exists, the amplification reaction using primer R3c is the signal of the SMN2 exon, and the SMN1-Target signal peak of the amplification reaction with R3 and R3b represents the non-specific amplification signal of the SMN2 exon.

[0200] The results are shown in Table 12 and Figure 2 . The results show that there will still be a certain degree of SMN2 gene amplification when only blocking primers are used. The combination of R3 primers and blocking probes used in experimental group 1 can effectively inhibit SMN2 amplification, with an inhibition efficiency of 99.65%. Experimental group 4 only used blocking primer R3, and the inhibition efficiency of SMN2 gene amplification was 80.88%. Experimental group 2 used primer R3b and blocking probe combination, and the inhibition efficiency of SMN2 gene amplification was 70.72%. Experimental group 5, using primer R3b alone, had an inhibition efficiency of 50.98% on SMN2 gene amplification. It can be seen that the blocking primers and blocking probes used in the present invention can effectively eliminate the interference of SMN2 gene amplification and improve the specificity and accuracy of detection.

[0201] Table 12

[0202]

[0203] Example 3: Screening of point mutation detection probe systems

[0204] A control probe is set up, and several adjacent quenching probe groups are changed to self-quenching probes. The relevant information and nucleotide sequence of the control probe are shown in Table 13.

[0205] Table 13 Related information and nucleotide sequence of control probe

[0206]

[0207]

[0208] The composition of the control gene detection solution A for one reaction (total volume of 6 μl) is shown in Table 14. The primers and probes in the gene detection solution A are used to detect c.22dupA, c.863G>T, c.400G>A, and c.463_464delAA. That is, the primers are the same as those used in Table 5, and the probes are replaced.

[0209] The composition of the control gene detection solution B for one reaction (total volume of 6 μl) is shown in Table 15. The primers and probes in the gene detection solution B are used to detect c.5C>G, c.40G>T, c.43C>T, c.56delT, c.835-5T>G, c.683T>A, and c.689C>T. That is, the primers are the same as those used in Table 6, and the probes are replaced.

[0210] Table 14

[0211]

[0212] Table 15

[0213]

[0214] The test sample is a normal newborn dried blood spot sample.

[0215] The kit prepared in step 3 of Example 1 was used and detection was performed according to step 4 of Example 1 (only gene detection solution A and gene detection solution B were used).

[0216] The gene detection solution A is replaced with the control gene detection solution A, and the gene detection solution B is replaced with the control gene detection solution B, and the detection is performed according to step 4 of Example 1.

[0217] Results Figure 3 , the signal intensity of the adjacent quenched probe is stronger than that of the self-quenched probe.

[0218] Example 4 Screening of detection probe systems for exon 7 and exon 8 of the SMN1 gene

[0219] A control probe was set up, and the adjacent quenching probe group and the self-quenching probe were exchanged. The relevant information and nucleotide sequence of the control probe are shown in Table 16.

[0220] Table 16

[0221]

[0222] The composition of the gene detection solution C of the control group (1 reaction, total volume of 6 μl) is shown in Table 17. The primers and probes in the gene detection solution C are used to detect exon 7 and exon 8 of the SMN1 gene, as well as SMN1-Target and SMN1-Ref.

[0223] Table 17

[0224]

[0225] The composition of the gene detection solution C of the test group (1 reaction, total volume of 6 μl) is shown in Table 18.

[0226] Table 18

[0227]

[0228] The subjects were confirmed SMN1 double copy deletion subjects (patients), SMN1 single copy deletion subjects (carriers), and SMN1 normal subjects, representing 0 copy, 1 copy, and 2 copy samples of SMN1 gene exon 7, respectively. The sample template solution was prepared by adding 100 μl of pretreatment solution to 3 μl of the subject's whole blood, incubating at 95°C for 10 min, and then centrifuging at 2500 rpm for 5 min, and collecting the supernatant, which was the sample template solution.

[0229] Replace the gene detection solution C, and the rest is the same as step 3 of Example 1. Mix 6.0 μl of amplification reaction solution, 2 μl of sample template solution and gene detection solution C, add deionized water to 25 μl, and centrifuge to remove bubbles to obtain the sample detection reaction system. Perform detection according to step 4 of Example 1, step 3.

[0230] The results are shown in Figure 4 and Figure 5 , SMN1 exon 7, the adjacent quenching dual probe system signal is stronger than the self-quenching system probe fluorescence signal, and the distinction effect of copy number 0, 1, and 2 is similar. SMN1 exon 8, the dual probe system signal is similar to the self-quenching system probe fluorescence signal, but the adjacent quenching dual probe system is significantly better than the self-quenching probe system in distinguishing the exon 8 copy number 1 and 2. For the SMN1-Target region, the self-quenching probe system probe signal is stronger than the adjacent dual probe system. The results are shown in Figure 6 .

[0231] Example 5: Plasmid Detection

[0232] This example involves 11 plasmids, namely plasmids Z1 to Z11. The plasmid-related information is shown in Table 19, which contains the wild-type or mutant gene sequence of the SMN1 gene locus to be tested. The plasmids were prepared by Sangon Biotech (Shanghai) Co., Ltd.: DNA molecules corresponding to each gene locus were synthesized by gene synthesis, and then cloned into the PUC-57 plasmid.

[0233] Table 19 Gene synthesis containing each SMN1 gene site sequence plasmid

[0234] Plasmids genotype The location of the foreign DNA molecule used to insert the plasmid in the genome (2013, GRCh38 / hg38) Z1 c.22dup (p.Ser8LysfsX23A) homozygous mutation Chr5: 70924941-70925480, 70925125 inserted in A Z2 c.683T>A (p.Leu228X) homozygous mutation Chr5: 70944501-70944980, base T at 70944713 mutated to A Z3 c.689C>T (p.Ser230Leu) homozygous mutation Chr5: 70944501-70944980, base C at 70944719 mutated to T Z4 c.863G>T (p.Gly279Glufs*5) homozygous mutation Chr5: Base G at 70951701-70952190, 70951969 mutated to T Z5 c.400G>A(p.Glu134Lys) homozygous mutation Chr5: Base G at 70942271-70942760, 70942482 mutated to A Z6 c.835-5T>G (p.Gly279Glufs*5) homozygous mutation Chr5: 70951701-70952190, 70951936 base T mutated to G Z7 c.463_464delAA (p.Asn155Cysfs*5) homozygous mutation Chr5: AA deletion at 70942271-70942760, 70942547-70942548 Z8 c.5C>G (p.Ala2Gly) homozygous mutation Chr5: 70924941-70925480, base C at 70925108 mutated to G Z9 c.40G>T (p.Glu13X) homozygous mutation Chr5: 70924941-70925480, base G at 70925143 mutated to T Z10 c.43C>T (p.Glu14X) homozygous mutation Chr5: 70924941-70925480, base C at 70925146 mutated to T Z11 c.56delT (p.Val19Glyfs*21) homozygous mutation Chr5: 70924941-70925480, base T is missing at 70925159

[0235] Take a plasmid (any one of plasmids Z1 to Z11) and dilute it to 0.1 pg / μl to prepare a simulated homozygous mutation sample.

[0236] There are 11 plasmids, and 11 simulated homozygous mutation samples were obtained accordingly.

[0237] The genomic DNA of normal people (sequencing verification has been performed and all relevant sites are wild type) was used as a wild sample.

[0238] The kit prepared in step 3 of Example 1 was used and detection was performed according to step 4 of Example 1 (only gene detection solution A and gene detection solution B were used).

[0239] Results Figure 7 and Figure 8 For the convenience of comparison, each figure is an overlay of simulated homozygous mutant samples (homozygous mutant type) and wild samples (wild type). The results show that the kit provided by the present invention can effectively detect the wild type and homozygous mutant type of the gene locus to be tested.

[0240] Example 6: Sensitivity test

[0241] Extract genomic DNA from normal people (sequencing verification has been performed, and all relevant sites are wild-type). Dilute the genomic DNA in a gradient manner and use it as a sample template solution.

[0242] The kit prepared in step 3 of Example 1 was used and detection was performed according to step 4 of Example 1. The amount of genomic DNA added as a template in each detection reaction system was set to 10 ng, 5 ng, 2.5 ng, 1.25 ng, 0.675 ng, 0.338 ng or 0.168 ng, respectively.

[0243] The results showed that the signal was still good even when the addition amount was as low as 1.25 ng. Therefore, the detection sensitivity was set to 2 ng / 25 μl reaction system to ensure the quality of the detection signal.

[0244] Example 7: Clinical sample verification

[0245] The subjects of this example are 16 patients who have been diagnosed with spinal muscular atrophy.

[0246] The subject collects anticoagulated blood to prepare a dried blood spot sample. The test kit prepared in step 3 of Example 1 is used and the test is performed according to step 4 of Example 1.

[0247] Anticoagulated blood was collected from the subjects, and blood DNA was purified using the Tiangen Genomic Purification Kit for MLPA verification and Sanger sequencing verification.

[0248] Among 16 clinical spinal muscular atrophy samples, 13 were detected to be caused by biallelic homozygous deletion of exon 7 of the SMN1 gene, and 3 were detected to be caused by compound heterozygous mutations of the SMN1 gene, that is, one allele was deleted and the other allele had a minor pathogenic variation (including point mutation).

[0249] The test results of the kit were consistent with the MLPA and sequencing verification results.

[0250] Example 8: Clinical sample verification

[0251] A large number of clinical subjects were tested to provide exemplary test results for various genotypes.

[0252] The subject collects anticoagulated blood to prepare a dried blood spot sample. The test kit prepared in step 3 of Example 1 is used and the test is performed according to step 4 of Example 1.

[0253] Anticoagulated blood was collected from the subjects, and blood DNA was purified using the Tiangen Genomic Purification Kit for MLPA verification and Sanger sequencing verification.

[0254] The kit can detect patients with biallelic homozygous deletion of exon 7 of the SMN1 gene. The exemplary detection effect diagram is shown in Fig. 9 In the FAM fluorescence channel of detection system C, there is no signal for the melting peak representing SMN1-exon7, and only the melting peak of SMN2-exon7 appears. In the ROX fluorescence channel, a melting peak appears for SMN1-Ref but no melting peak signal for SMN1-Target.

[0255] The kit can detect carriers of SMN1 gene exon 7 single copy deletion. See the exemplary detection effect diagram Fig.10 .

[0256] The kit can detect normal samples (double copies of SMN1 gene exon 7). See the exemplary detection effect diagram. Fig.11 .

[0257] In the ROX fluorescence channel of detection system C, the melting peak ratio of SMN1-Target and SMN1-Ref decreased. The quantitative calculation is shown in Fig.12The melting peak ratio of SMN1-Target and SMN1-Ref was used to detect whether the copy number of SMN1 exon 7 was 0, 1 or 2. Different copy numbers of SMN1 exon 7 (0, 1, 2) could be distinguished well. When a single copy of SMN1 was deleted (carrier), the intensity ratio of the high Tm melting peak and the low Tm melting peak changed. Rm(SMN1-Target) was the intensity of the high Tm melting peak, and Rm(SMN1-Ref) was the intensity of the low Tm melting peak. ROX Ratio=Rm(SMN1-Target) / Rm(SMN1-Ref); when 0.75≤ROX Ratio≤1.05, the copy number of SMN1 exon 7 region was 1 copy; when 1.45≥ROX Ratio≥1.2, the copy number of SMN1 exon 7 region was 2.

[0258] The kit can detect patients with compound heterozygous mutations of the SMN1 gene. The deletion of one allele is determined by the ROX fluorescence channel in the detection system C, and the melting peak ratio of the carrier SMN1-Target to SMN1-Ref is reduced. The detection of allele point mutations is detected by the detection reaction system A and B. Three patients with compound heterozygous mutations are detected to carry common point mutations: c.22dup (p.Ser8LysfsX23A), c.400G>A (p.Glu134Lys), and c.863G>T (p.Gly279Glufs*5). The exemplary results are shown in Fig.13 , Fig.14 , Fig.15 For ease of comparison, each figure is an overlay of exemplary samples.

[0259] The kit can accurately detect SMN1 complete deletion samples and single copy deletion samples (carriers), and investigates the interference of different common copy numbers of SMN2 exon 7 on the detection of SMN1 exon 7 copy number. Fig.16 The common copy numbers of SMN2 exon 7 are 0, 1, 2, and 3 copies, which do not affect the detection results of SMN1 exon 7 copy number. This kit has good anti-interference ability for SMN2.

[0260] The test results of the kit were consistent with the MLPA and sequencing verification results.

[0261] Example 9: Dried blood spot sample detection

[0262] The samples to be tested in this embodiment are 80 dried blood spot samples of newborns.

[0263] The kit prepared in step 3 of Example 1 was used and the detection was performed according to step 4 of Example 1.

[0264] Test results: No SMN1 exon 7 homozygous deletion mutation was found, and 2 cases of exon 7 heterozygous deletion (carriers) were found.

[0265] Example 10: Saliva sample detection

[0266] The samples to be tested in this embodiment are 10 human saliva samples.

[0267] The kit prepared in step 3 of Example 1 was used and the detection was performed according to step 4 of Example 1.

[0268] The SMN1 gene detection kit prepared in Example 1 was used to detect the sample to be tested.

[0269] Test results: All signals were detected, and the detection signals were good. No SMN1 exon 7 homozygous deletion mutation or heterozygous deletion (carrier) was found.

[0270] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A kit comprising gene detection liquid A, gene detection liquid B, and gene detection liquid C; The gene detection solution A contains primer F4, primer R4, primer F6, primer R6, primer F7, primer R7, probe FP4, probe Q2, probe FP8, probe Q5, probe FP9, probe FP10 and probe Q6; Gene detection solution B contains primer F4, primer R4, primer F6, primer R6, primer F5, primer R5, probe FP6, probe Q4, probe FP7, probe FP11, probe Q7, probe FP5 and probe Q3; The gene detection solution C contains primer F1, primer R1, primer F2, primer R2, primer F3, primer R3, probe FP1, probe FP2, probe Q1, probe FP3 and probe FB1; Primer F1 is the single-stranded DNA molecule shown in SEQ ID NO: 1; primer R1 is the single-stranded DNA molecule shown in SEQ ID NO: 2; primer F2 is the single-stranded DNA molecule shown in SEQ ID NO: 3; primer R2 is the single-stranded DNA molecule shown in SEQ ID NO: 4; primer F3 is the single-stranded DNA molecule shown in SEQ ID NO: 5; primer R3 is the single-stranded DNA molecule shown in SEQ ID NO: 6; primer F4 is the single-stranded DNA molecule shown in SEQ ID NO: 7; primer R4 is the single-stranded DNA molecule shown in SEQ ID NO: 8; primer F5 is the single-stranded DNA molecule shown in SEQ ID NO: 9; primer R5 is the single-stranded DNA molecule shown in SEQ ID NO: 10; primer F6 is the single-stranded DNA molecule shown in SEQ ID NO: 11; primer R6 is the single-stranded DNA molecule shown in SEQ ID NO: 12; primer F7 is the single-stranded DNA molecule shown in SEQ ID NO: 13; primer R7 is the single-stranded DNA molecule shown in SEQ ID NO: 14; Probe FP1 is a single-stranded DNA molecule shown in SEQ ID NO: 15; probe FP2 is a single-stranded DNA molecule shown in SEQ ID NO: 16; probe Q1 is a single-stranded DNA molecule shown in SEQ ID NO: 17; probe FP3 is a single-stranded DNA molecule shown in SEQ ID NO: 18; probe FB1 is a single-stranded DNA molecule shown in SEQ ID NO: 19; probe FP4 is a single-stranded DNA molecule shown in SEQ ID NO: 20; probe Q2 is a single-stranded DNA molecule shown in SEQ ID NO: 21; probe FP5 is a single-stranded DNA molecule shown in SEQ ID NO: 22; probe Q3 is a single-stranded DNA molecule shown in SEQ ID NO: 23; probe FP6 is a single-stranded DNA molecule shown in SEQ ID NO: The probe is the single-stranded DNA molecule shown in SEQIDNO: 24; the probe Q4 is the single-stranded DNA molecule shown in SEQIDNO: 25; the probe FP7 is the single-stranded DNA molecule shown in SEQIDNO: 26; the probe FP8 is the single-stranded DNA molecule shown in SEQIDNO: 27; the probe Q5 is the single-stranded DNA molecule shown in SEQIDNO: 28; the probe FP9 is the single-stranded DNA molecule shown in SEQIDNO: 29; the probe Q6 is the single-stranded DNA molecule shown in SEQIDNO: 30; the probe FP10 is the single-stranded DNA molecule shown in SEQIDNO: 31; the probe FP11 is the single-stranded DNA molecule shown in SEQIDNO: 32; the probe Q7 is the single-stranded DNA molecule shown in SEQIDNO:

33.

2. The kit according to claim 1, characterized in that: The kit also includes a pre-treatment solution and an amplification reaction solution; The pretreatment solution contains Chelex-100, Triton-X100 and NaOH; The amplification reaction solution contains DNA polymerase, dNTP, dUTP and UDG enzyme.

3. The kit according to claim 1 or 2, characterized in that: In gene detection solution A, the molar ratios of primer F4, primer R4, primer F6, primer R6, primer F7, primer R7, probe FP4, probe Q2, probe FP8, probe Q5, probe FP9, probe FP10 and probe Q6 are as follows: 1.5:15:12:1.2:1:10:6:6:6:6:5:5:5; In gene detection solution B, the molar ratios of primer F4, primer R4, primer F6, primer R6, primer F5, primer R5, probe FP6, probe Q4, probe FP7, probe FP11, probe Q7, probe FP5 and probe Q3 are as follows: 1.5：15：1：10：1.5：15：7.5：7.5：7.5：5：5：6：6； In gene detection solution C, the molar ratios of primer F1, primer R1, primer F2, primer R2, primer F3, primer R3, probe FP1, probe FP2, probe Q1, probe FP3, and probe FB1 are as follows: 1:10:1:10:10:1:5:5:5:5:

2.

4. The kit according to claim 1 or 2, characterized in that: Probe FP1, 5′ end labeled with FAM and 3′ end labeled with BHQ2; Probe FP2, 5′ end labeled with CY5, 3′ end modified with C3Spacer; Probe Q1, 3′ end labeled with BHQ2; Probe FP3, 5′ end labeled with ROX and 3′ end labeled with BHQ2; Probe FB1, the 12th and 13th nucleotides were modified with locked nucleic acid, and the 3′ end was modified with C3Spacer; Probe FP4, 5′ end labeled with CY5, 3′ end modified with C3Spacer; Probe Q2, 3′ end labeled with BHQ2; Probe FP5, 5′ end labeled with ROX, 3′ end modified with C3Spacer; Probe Q3, 3′ end labeled with BHQ2; Probe FP6, 3′ end labeled with FAM; Probe Q4, 5′ end labeled with BHQ1 and 3′ end labeled with BHQ1; Probe FP7, 5′ end labeled with HEX, 3′ end modified with C3Spacer; Probe FP8, 5′ end labeled with ROX, 3′ end modified with C3Spacer; Probe Q5, 3′ end labeled with MGB; Probe FP9, 3′ end labeled with FAM; Probe Q6, 5′ end labeled with BHQ1 and 3′ end labeled with BHQ1; Probe FP10, 5′ end labeled with HEX, 3′ end modified with C3Spacer; Probe FP11, 5′ end labeled with CY5, 3′ end modified with C3Spacer; Probe Q7, 3' end labeled with MGB.

5. Application of gene detection solution A, gene detection solution B and gene detection solution C in preparing a kit; The gene detection liquid A is the gene detection liquid A described in claim 1; The gene detection liquid B is the gene detection liquid B described in claim 1; The gene testing solution C is the gene testing solution C described in claim 1.

6. Application of gene detection solution A, gene detection solution B, gene detection solution C, pretreatment solution and amplification reaction solution in preparing a kit; The gene detection liquid A is the gene detection liquid A described in claim 1; The gene detection liquid B is the gene detection liquid B described in claim 1; The gene detection liquid C is the gene detection liquid C described in claim 1; The pretreatment liquid is the pretreatment liquid described in claim 2; The amplification reaction solution is the amplification reaction solution described in claim 2.

7. The use according to claim 5 or 6, characterized in that: In gene detection solution A, the molar ratios of primer F4, primer R4, primer F6, primer R6, primer F7, primer R7, probe FP4, probe Q2, probe FP8, probe Q5, probe FP9, probe FP10 and probe Q6 are as follows: 1.5:15:12:1.2:1:10:6:6:6:6:5:5:5; In gene detection solution B, the molar ratios of primer F4, primer R4, primer F6, primer R6, primer F5, primer R5, probe FP6, probe Q4, probe FP7, probe FP11, probe Q7, probe FP5 and probe Q3 are as follows: 1.5：15：1：10：1.5：15：7.5：7.5：7.5：5：5：6：6； In gene detection solution C, the molar ratios of primer F1, primer R1, primer F2, primer R2, primer F3, primer R3, probe FP1, probe FP2, probe Q1, probe FP3, and probe FB1 are as follows: 1:10:1:10:10:1:5:5:5:5:

2.

8. The use according to claim 5 or 6, characterized in that: Probe FP1, 5′ end labeled with FAM and 3′ end labeled with BHQ2; Probe FP2, 5′ end labeled with CY5, 3′ end modified with C3Spacer; Probe Q1, 3′ end labeled with BHQ2; Probe FP3, 5′ end labeled with ROX and 3′ end labeled with BHQ2; Probe FB1, the 12th and 13th nucleotides were modified with locked nucleic acid, and the 3′ end was modified with C3Spacer; Probe FP4, 5′ end labeled with CY5, 3′ end modified with C3Spacer; Probe Q2, 3′ end labeled with BHQ2; Probe FP5, 5′ end labeled with ROX, 3′ end modified with C3Spacer; Probe Q3, 3′ end labeled with BHQ2; Probe FP6, 3′ end labeled with FAM; Probe Q4, 5′ end labeled with BHQ1 and 3′ end labeled with BHQ1; Probe FP7, 5′ end labeled with HEX, 3′ end modified with C3Spacer; Probe FP8, 5′ end labeled with ROX, 3′ end modified with C3Spacer; Probe Q5, 3′ end labeled with MGB; Probe FP9, 3′ end labeled with FAM; Probe Q6, 5′ end labeled with BHQ1 and 3′ end labeled with BHQ1; Probe FP10, 5′ end labeled with HEX, 3′ end modified with C3Spacer; Probe FP11, 5′ end labeled with CY5, 3′ end modified with C3Spacer; Probe Q7, 3' end labeled with MGB.

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