Primer group and kit for detecting multiple mutations of spinal muscular atrophy and application of primer group and kit

Abstract

The invention provides a primer group and a kit for detecting multiple mutations of spinal muscular atrophy and application of the primer group and the kit. Specifically, the invention provides a primer group, and the primer group comprises an SMA-1F / 1R primer pair, an SMA-2F / 2R primer pair and a reference gene primer pair. The invention also provides a kit which comprises the primer group. Based on long-fragment multiplex PCR amplification and single-molecule gene sequencing, multiple mutations related to SMN1 / SMN2 of SMA pathogenic genes of multiple samples can be detected at the same time in a high-specificity, accurate and rapid manner.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a method for detecting spinal muscular atrophy by using a single molecule gene sequencing platform through haplotype and internal reference-standard quantitative analysis. SMN1 / SMN2 Primer sets and kits for multiple gene mutations and their applications. Background Art

[0002] Spinal muscular atrophy (SMA) is a group of diseases caused by SMN1 Autosomal recessive genetic disease caused by homozygous gene deletion 1 . Clinical manifestations are progressive muscle weakness and atrophy, accompanied by spinal cord degeneration, and in the most severe cases, damage to the lower motor neurons. SMA is one of the leading genetic causes of infant mortality and is found in almost all populations. According to statistics, the incidence of SMA is 1 in every 6,000-11,000 live births, and the incidence of type I SMA is 4.1 in every 100,000 live births. In the general population SMN1 The mutation is carried by 2% to 3% (1 in 40) 2 Despite the high population carrier frequency, the incidence of SMA is lower than expected. This may reflect that some fetuses have 0 / 0 SMN1 / SMN2 genotype (i.e., the absence of SMN protein at all), resulting in embryonic lethality.

[0003] The natural history and findings of SMA depend on phenotypic variation and are clinically divided into SMA "types." Of all the SMA types, five are outlined below: Type 0 SMA presents in the neonatal period, with death typically occurring at birth or within the first month of life; Type I SMA is characterized by severe generalized muscle weakness and hypotonia that develops at birth or within the first three months. Death from respiratory failure typically occurs within the first two years; Children with Type II SMA are able to sit, but they cannot stand or walk independently and typically survive for more than four years; Type III SMA is a milder form that develops in infancy or young adulthood, with Type III SMA patients learning to walk independently and having a longer survival rate; Type IV SMA, patients present in adulthood and are the mildest phenotype of SMA 3 .

[0004] Survival motor neurons (SMN) The gene consists of nine exons, about 28 kb in length, and there are two almost identical SMN Gene: SMN1 Gene and SMN2 Gene 4 .because SMN2A critical single nucleotide mutation occurs in exon 7 of SMN2 The gene produces less functional SMN protein, just SMN1 10-15% 5 About 95% of people with SMA have SMN1 Homozygous deletion of the gene, while 5% have compound heterozygous mutations. Most SMA patients have SMN1 Homozygous deletion of exon 7 of the gene, and is dependent on residual SMN2 Produce functional SMN protein. Therefore, SMN2 There is a positive correlation between the number of copies of the gene and the severity of the phenotype, with most patients with severe type I having one or two copies of the gene. SMN2 copies; most people with type II have 3 SMN2 copies; and most people with type III have 3 or 4 SMN2 Copy. In normal people SMN2 The number of copies of the gene varies from zero to three, with approximately 10%–15% of the population having none. SMN2 6 .

[0005] Currently, the most commonly used method for molecular detection of SMA is to detect large fragment deletions or copy number variations through MLPA or qPCR, combined with Sanger sequencing specific detection SMN1 Point mutations on genes. Since the copy number of housekeeping genes is stable, traditional methods such as qPCR and MLPA use the housekeeping gene sequence to design a small fragment internal reference, and perform relative quantification of the copy number of exons 7 and 8 of the SMN1 gene to determine whether a deletion mutation has occurred. However, qPCR and MLPA can only detect the gene copy number based on the gene copy number. SMN1 The absence of SMN1 Point mutation 7 About 5% of SMA patients carry a point mutation in SMN1 Therefore, some SMA patients are inevitably misdiagnosed as carriers. SMN1 and SMN2 The high homology of SMN2 It will also exist SMN1 The above characteristic sequences will also interfere with the specificity of PCR amplification primers and sequencing primers during Sanger sequencing, leading to missed diagnosis and misdiagnosis in actual testing. At the same time, conventional diagnostic methods do not distinguish SMN1 The missing and SMN1 Whether SMN2 Homologous recombination occurs.

[0006] Next-generation sequencing (NGS) requires SMN1 and SMN2Separately construct NGS sequencing libraries to enable specific detection SMN1 Gene or SMN2 point mutations, is not compatible with other gene tests, and the process is cumbersome 8 . Currently published research SMN Methods for complete gene sequence include whole genome sequencing (WGS) and multiplexed direct genomic selection (MDiGS) sequencing. WGS is a fairly expensive and laborious technique that often requires complex bioinformatics analysis. In MDiGS, the entire DNA is prepared in libraries, and the experimental steps are also quite complex.

[0007] Single-molecule gene sequencing does not require PCR amplification, and can sequence each DNA molecule individually. It has the characteristics of ultra-long read length (read length can reach 30 kb-100 kb), high accuracy, no GC preference and single-molecule resolution, and can achieve accurate detection of various gene variation information. 9 Li et al. 10 Amplification by LR-PCR SMN1 and SMN2 The full-length sequences of the genes were used to determine the copy numbers of SMN1 and SMN2 in 337 samples, but the reliance on haplotype analysis made it difficult to call the exact copy number in rare cases when both SMN1 and SMN2 had only one haplotype and a 1:1 read ratio.

[0008] Currently, methods based on qPCR, MLPA, Sanger sequencing or second-generation and single-molecule gene sequencing can achieve SMN1 and SMN2 Gene copy number and partial point mutation detection are detected, but the detection has the following main limitations: 1. It is impossible to achieve simultaneous detection in the same system SMN1 and SMN2 Detection of all point mutation types and copy number variations of genes; 2. Due to SMN1 and SMN2 There is a possibility of multiple recombination, which affects the binding of MLPA probes and the design of Sanger sequencing primers, resulting in a certain degree of missed detection and false detection; 3. For SMN1 and SMN2 The copy number detection judgment is not accurate enough; 4. When two or more mutations exist at the same gene locus, existing methods cannot distinguish between cis or trans mutations, and whether the mutations have a linkage effect. Summary of the invention

[0009] In view of this, the present invention provides a method based on long-fragment multiplex PCR amplification and single-molecule gene sequencing to detect multiple mutations related to SMA. SMN1 / SMN2 and long fragment internal reference amplification; combined with the long read length of the single-molecule gene sequencing platform, it is used to detect SMN1 / SMN2 The copy number of the gene and the point mutation within the detection range, in order to improve the accuracy of the copy number and detect the situation that SMN1 and SMN2 have only one haplotype and the reading ratio is 1:1, we designed the primer combination based on the characteristics of the stable copy number of the housekeeping gene and the long read length of the single-molecule sequencing platform, and provided a combination of haplotype analysis and internal reference-standard quantitative analysis to achieve accurate copy number judgment, which can accurately, quickly and high-throughput detect SMA-related gene mutations. The method involved in the present invention is simple to operate, and the quality of long-fragment multiplex PCR and single-molecule sequencing libraries is reliable and reproducible, which is conducive to the application of single-molecule gene sequencing technology in clinical detection.

[0010] The purpose of the present invention is to solve the problem of SMA pathogenic genes at present SMN1 / SMN2 The detection is cumbersome, the specific NGS detection process misses the detection, the NGS detection cannot distinguish the cis-trans structure of the mutation, and the single-molecule sequencing platform SMN1 and SMN2 have only one haplotype and the reading ratio is 1:1, and the copy number cannot be accurately determined. SMN1 / SMN2 The long-fragment internal reference amplification and preparation of single-molecule sequencing libraries can achieve the goal of comprehensive, accurate and rapid detection of multiple SMA mutations in multiple samples.

[0011] The prior art is based only on SMN1 and SMN2 Haplotype reads ratio to determine SMN1 and SMN2 For example, when SMN1 and SMN2 When the haplotype is 1 and the number of reads is 1200, the existing method will convert SMN1 and SMN2 The copy number of each of the primers, kits and methods of the present invention can be determined as 1 copy. SMN1 and SMN2 The relationship between the haplotype and the read ratio is further determined when the haplotype is 1. The primer set, kit and method of the present invention can be used to detect the copy number by the following steps: First, haplotype analysis is performed to distinguish SMN1 and SMN2 Genes are counted and the reads of each haplotype are counted; then the internal reference-standard is used to correct the SMN1 andSMN2 The amplification deviation between SMN1 and SMN2 The reads ratio between the sample and the internal reference is used to finally determine the copy number of the sample to be tested.

[0012] In a first aspect, the present invention provides a primer set for simultaneously detecting multiple mutations of an SMA pathogenic gene, comprising an SMA-1F / 1R primer pair, an SMA-2F / 2R primer pair and an internal reference gene primer pair; wherein the internal reference gene primer pair is selected from a B2M-F / R primer pair, a GAPDH-F / R primer pair, an ACTB-F / R primer pair or an SDHA-F / R primer pair; wherein the nucleotide sequence of the SMA-1F primer is as shown in any one of SEQ ID NOs: 1-3, and the nucleotide sequence of the SMA-1R primer is as shown in any one of SEQ ID NOs: 4-6; wherein the nucleotide sequence of the SMA-2F primer is as shown in any one of SEQ ID NOs: 7-9, and the nucleotide sequence of the SMA-2R primer is as shown in any one of SEQ ID NOs: 10-12; wherein the nucleotide sequence of the B2M-F primer is as shown in any one of SEQ ID NOs: 13-15, and the nucleotide sequence of the B2M-R primer is as shown in SEQ ID NOs: 16-18; wherein the nucleotide sequence of the GAPDH-F primer is shown in any one of SEQ ID NOs: 19-21, wherein the nucleotide sequence of the GAPDH-R primer is shown in any one of SEQ ID NOs: 22-24; wherein the nucleotide sequence of the ACTB-F primer is shown in SEQ ID NO: 25 or 26, wherein the nucleotide sequence of the ACTB-R primer is shown in SEQ ID NO: 27 or 28; wherein the nucleotide sequence of the SDHA-F primer is shown in SEQ ID NO: 29 or 30, wherein the nucleotide sequence of the SDHA-R primer is shown in SEQ ID NO: 31 or 32.

[0013] The primer set of the present invention comprises a pair of SMA pathogenic gene amplification primers SMA-1F / 1R and SMA-2F / 2R and a pair of long fragment internal reference amplification primers GAPDH-F / R, ACTB-F / R, B2M-F / R or SDHA-F / R (primer positions are as shown in FIG. Figure 1 The above primer pair can amplify the primer range on the genome. SMN1 / SMN2 The complete sequences of the genes and reference genes, including any type of mutation sequences within the primer range.

[0014] In some preferred embodiments, the amplification product of the SMA-1F / 1R primer pair, the SMA-2F / 2R primer pair and the internal reference gene primer pair is about 15 kb. In some preferred embodiments, if there is a SNP at the primer position, a degenerate base primer is used.

[0015] In some embodiments, the primer set described in the present invention is used to simultaneously detect SMN1 Genes and SMN2 In some embodiments, the primer set is used to simultaneously amplify SMN1 Gene, SMN2 In some preferred experimental schemes, the primer set is used for long fragment multiplex PCR amplification in a system SMN1 / SMN2 2 gene fragments and 1 reference gene fragment.

[0016] Therefore, in some embodiments, the primer set is used to simultaneously detect SMN1 Genes and SMN2 The primer set of the present invention can be used for PCR amplification of a system including SMA-related pathogenic genes. SMN1 / SMN2 The fragments of various mutation types and internal reference gene fragments within the range can be combined with the subsequent PacBio or Nanopore sequencing platform to detect SMN1 / SMN2 All mutation types of gene fragments. In some embodiments, the copy number is detected by haplotype and internal reference-standard quantitative analysis.

[0017] In some preferred embodiments, the point mutations include at least 168 point mutations as shown in Table 1. In some preferred embodiments, the detection of copy number variation includes SMN1 Genes and SMN2 The copy number was detected when the gene had only one haplotype and the read ratio was 1:1.

[0018]

[0019]

[0020]

[0021]

[0022] In some embodiments, the 5' end of the primer of the primer set also contains a DNA barcode for distinguishing different samples. In some preferred embodiments, the 5' end barcodes of the F and R primers can be the same or different, and those skilled in the art can choose as needed. In some preferred embodiments, the length of the DNA barcode is 5-50nt.

[0023] The second aspect of the present invention provides a kit for simultaneously detecting multiple mutations of SMA pathogenic genes, which comprises the following reagents: (1) reagents for multiplex PCR amplification; (2) internal reference gene standards for quantitative analysis; (3) reagents for constructing a single molecule sequencing library; wherein the reagents for multiplex PCR amplification comprise the primer set described in the present invention.

[0024] In some embodiments, the kit is used to simultaneously detect SMN1 Genes and SMN2 The primer set of the present invention can be used for PCR amplification of a system including SMA-related pathogenic genes. SMN1 / SMN2 The kit of the present invention can detect fragments of various mutation types and internal reference gene fragments within the range, and then combined with the subsequent PacBio or Nanopore sequencing platform. SMN1 / SMN2 In some preferred embodiments, the detection of copy number variation is included in SMN1 Genes and SMN2 The copy number is detected when the gene has only one haplotype and the read ratio is 1: 1. In some preferred embodiments, the point mutations include at least 168 point mutations as shown in Table 1.

[0025] In some embodiments, the reagents for multiplex PCR amplification further comprise a DNA polymerase and a reaction buffer. In some embodiments, the reagents for multiplex PCR amplification are used to simultaneously amplify in one reaction system SMN1 Gene, SMN2 For the kit, the PCR amplification product may be purified or not before the next step of reaction, and those skilled in the art may choose according to the need.

[0026] In some embodiments, the reagents for constructing a single-molecule sequencing library include an adapter, a ligase, DNA purification magnetic beads, a reaction buffer, and an exonuclease.

[0027] In some preferred embodiments, the kit can simultaneously detect at least SMN1 / SMN2Gene point mutation and copy number detection by haplotype and internal reference-standard based quantitative analysis. In a specific embodiment, for the kit, long fragment multiplex PCR amplification is completed in one reaction tube.

[0028] The third aspect of the present invention provides a system for simultaneously detecting multiple mutations in SMA pathogenic genes, which comprises the following modules: (1) an acquisition module: used to obtain and prepare a sample from a subject; (2) an amplification module: used to perform multiple PCR amplification on the sample; (3) a library construction module: used to construct a single-molecule sequencing library; (4) a sequencing module: used to sequence and analyze the mutation type; wherein the multiple PCR amplification in the acquisition module is performed using the primer set described in the present invention or the kit described in the present invention.

[0029] In some embodiments, the multiple mutations in the SMA pathogenic gene include SMN1 Genes and SMN2 The primer set of the present invention can be used for PCR amplification of a system including SMA-related pathogenic genes. SMN1 / SMN2 The system provided by the present invention can detect fragments of various mutation types and internal reference gene fragments within the range, and then combined with the subsequent PacBio or Nanopore sequencing platform. SMN1 / SMN2 In some preferred embodiments, the detection of copy number variation is included in SMN1 Genes and SMN2 The copy number is detected when the gene has only one haplotype and the read ratio is 1: 1. In some preferred embodiments, the point mutations include at least 168 point mutations as shown in Table 1.

[0030] In some embodiments, the sample is selected from a biological sample or gDNA extracted from the biological sample. In some preferred embodiments, the biological sample comprises cultured cell lines, blood, amniotic fluid, chorionic villi, gametes, blastocyst cells, joint fluid, urine, sweat, saliva, feces, cerebrospinal fluid, ascites, pleural effusion, bile and pancreatic fluid.

[0031] In some embodiments, the sequencing module is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

[0032] In a preferred embodiment, the single-molecule gene sequencing is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

[0033] In a specific embodiment, PacBio library adapter ligation can be performed using blunt-end ligation or TA ligation.

[0034] In a specific embodiment, the PacBio universal blunt-end adapter sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3' (SEQ ID NO: 33), which is annealed to form a blunt-end stem-loop structure adapter adapter. DNA (barcode) with different sequences of 5-50nt can be added to the stem to form adapter adapters with different barcodes. PacBio libraries with different barcodes can be mixed together for sequencing.

[0035] In a specific embodiment, the PacBio universal TA adapter sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 34), and a blunt-ended stem-loop structure adapter adapter is formed by annealing. 5-50nt of different sequence DNA (Barcode) can be added to the stem to form adapter adapters with different Barcodes. PacBio libraries with different Barcodes can be mixed together for sequencing.

[0036] In one embodiment, the PacBio connector may or may not have a barcode. Preferably, the PacBio connector has a barcode designed by PacBio or a barcode designed by the company itself, and those skilled in the art can choose according to their needs.

[0037] In a preferred embodiment, the PacBio library is compatible with the Pacific Biosciences sequencing platform.

[0038] In a preferred embodiment, the reagents used to construct the Nanopore library include end repair enzymes, linkers, ligases, DNA purification magnetic beads, 80% ethanol and reaction buffer.

[0039] In one embodiment, Nanopore library adapter ligation can be performed using blunt-end ligation or TA ligation.

[0040] In one embodiment, the Nanopore connector may or may not have a barcode. Preferably, the Nanopore connector has a barcode designed by ONT or a self-designed barcode, and those skilled in the art can select one according to their needs.

[0041] In a preferred embodiment, the Nanopore library matches the sequencing platform of ONT.

[0042] Therefore, the excellent technical effects of the primer set, kit, system and method provided by the present invention mainly lie in the following aspects: (1) Wide detection range. The present invention can simultaneously detect the SMA-related genes that have been discovered in research. SMN1 There are 168 point mutations in total (as listed in Table 1), as well as SMN1 and SMN2 Unknown type of point mutation within the upper primer range; can detect SMN1 and SMN2 Deletions within the gene primer range and detection by haplotype and internal reference-based quantitative analysis SMN1 and SMN2 Copy number variation: Due to the read length measurement characteristics of single-molecule gene sequencing, the method of the present invention can also detect whether different mutations are linked.

[0043] (2) Detection of multiple mutation types with a single kit. Traditional methods require a detection system to be set up for each mutation type, while the present invention can simultaneously detect multiple mutations, including SNV, Indel, gene recombination, and copy number variation, in one reaction primer system.

[0044] (3) Multiple copy number analysis methods. In order to improve the accuracy of copy number, we designed a long fragment internal reference as a reference, and provided a method based on haplotype analysis and internal reference-standard quantitative analysis to achieve accurate copy number judgment.

[0045] (3) Low false positive and missed detection rates. Currently, the most common detection of SMA pathogenic genes SMN1 and SMN2 The copy number method is MLPA or qPCR. Since approximately 5% of SMA patients carry a point mutation SMN1 Therefore, some SMA patients are inevitably misdiagnosed as carriers, resulting in false negative judgments. The method used in the present invention directly and specifically amplifies SMN1 and SMN2 There is no risk of misdiagnosis or missed diagnosis of patients because the gene is only detected by the test.

[0046] (4) Sample diversification. The template used for PCR can be peripheral blood, dried blood spots or extracted genomic DNA, or human cell lines or other specific tissues.

[0047] (5) High-throughput detection. Single-molecule gene sequencing can achieve 384 types of barcode adapters, and more types of barcode adapters can be designed according to needs. Or a dual barcode system with primers and adapters can be used to achieve more barcode combinations. The high-throughput characteristics of the single-molecule gene sequencing platform determine that high-throughput sample detection can be achieved.

[0048] (6) High accuracy. PacBio's dumbbell-shaped library can be read multiple times during sequencing, and the base accuracy of the sequencing results after correction is greater than 99%. In addition, PacBio sequencing errors are random, and the base accuracy is greater than 99.9% after correction through sequencing depth. Therefore, gene mutations within the primer detection range can be accurately read.

[0049] (7) Flexible detection time. The Nanopore platform can generate data within minutes, and data analysis can be started within minutes or hours depending on the actual data volume. When the detection timeliness requirements are relatively high, the Nanopore platform has a time advantage. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a schematic diagram of PCR primer design.

[0051] Figure 2 Long-range PCR amplification SMN1 / SMN2 And DNA gel electrophoresis of long-fragment internal reference genes.

[0052] Figure 3 is a normal sample ( SMN1 - Two copies / SMN2 - two copies) of PacBio sequencing results.

[0053] Figure 4 is an SMA carrier sample ( SMN1 -One copy / SMN2 - two copies) of PacBio sequencing results.

[0054] Figure 5 This is the PacBio sequencing result of SMA patient samples (SMN1-zero copy / SMN2-two copies).

[0055] Figure 6 This is the PacBio sequencing result of the SMN1 point mutation sample. DETAILED DESCRIPTION

[0056] While describing in detail exemplary embodiments of the present invention, it should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0057] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope. Although any methods and materials similar or equivalent to those described herein can be used in the practice or test of the present disclosure, what is described below is preferred materials and methods.

[0058] The sequences used in the present invention are shown in Table 2.

[0059]

[0060] Example 1: Using the long-fragment multiplex PCR method of the present invention to amplify SMA-related pathogenic genes using different primer combinations SMN1 / SMN2 fragments and reference gene (B2M, GAPDH, ACTB or SDHA) fragments.

[0061] The primer set used in this example includes a SMA-1F / 1R primer pair, a SMA-2F / 2R primer pair, and an internal reference gene primer pair.

[0062] Wherein, for the SMA-1F / 1R primer pair, the nucleotide sequence of the SMA-1F primer is selected from any one of SEQ ID NOs: 1-3, and the nucleotide sequence of the SMA-1R primer is selected from any one of SEQ ID NOs: 4-6; for the SMA-2F / 2R primer pair, the nucleotide sequence of the SMA-2F primer is selected from any one of SEQ ID NOs: 7-9, and the nucleotide sequence of the SMA-2R primer is selected from any one of SEQ ID NOs: 10-12; for the B2M internal reference gene primer pair, the nucleotide sequence of the B2M-F primer is selected from any one of SEQ ID NOs: 13-15, and the nucleotide sequence of the B2M-R primer is selected from SEQ ID NOs: 16-18; for the GAPDHF internal reference gene primer pair, including GAPDHF1 / GAPDHR1 primer pair, GAPDHF2 / GAPDHR2 primer pair and GAPDHF3 / GAPDHR3 primer pair; for the ACTB internal reference gene primer pair, including ACTBF1 / ACTBR1 primer pair and ACTBF2 / ACTBR2 primer pair; for the SDHA internal reference gene primer pair, including SDHAF1 / SDHAR1 primer pair and SDHAF2 / SDHAR2 primer pair.

[0063] The peripheral blood, dried blood spots and genomic DNA samples were obtained and prepared using the collection module and the samples were amplified by long-fragment multiplex PCR using the amplification module. The reaction system was prepared according to Table 3 (long-fragment multiplex PCR) below.

[0064]

[0065] On the PCR instrument, pre-amplification was performed according to the conditions shown in Table 4 below:

[0066] After amplification, 5 mL of each sample was taken and tested on a 1% DNA gel. The results were as follows: Figure 2 As shown, different primer combinations effectively amplified the SMN1 gene, SMN2 gene and the internal reference gene.

[0067] Example 2: Construction of a PacBio sequencing library using the PCR method designed by the present invention

[0068] The reaction system was prepared according to Table 5 (long fragment multiplex PCR) below, and the SMA-related pathogenic genes of different mutation types were amplified using the primer combinations effectively amplified in Example 1. SMN1 / SMN2 Peripheral blood samples of the PCR fragments and internal reference fragments.

[0069]

[0070] On a PCR instrument, pre-amplification was performed according to the conditions shown in Table 6 below.

[0071]

[0072] After amplification, the amplified product was placed in a centrifuge at 10,000 rpm for 20 minutes. After centrifugation, the product was placed horizontally and 4 mL of supernatant was added to a new tube.

[0073] Step 2: Construction of PacBio sequencing library The single-molecule sequencing library was constructed using the library construction module, and the sequencing module was used to sequence and analyze the mutation type. The reaction system was prepared according to Table 7 below.

[0074]

[0075] The reaction was carried out in a PCR machine under the following conditions: 37ºC 20 min; 25ºC 15 min; 65ºC 10 min. After the reaction was completed, 0.5 mL Exonuclease III (NEB, Cat#M0206L) and 0.5 mL Exonuclease VII (NEB, Cat#M0379L) were added and the reaction was continued at 37ºC for 1 hour. The DNA was purified twice using 0.6x Ampure PB magnetic beads (PacBio, Cat#100-265-900) according to the manufacturer's instructions, and finally eluted with 10 mL Elution Buffer. The obtained DNA eluate is the target DNA PacBio sequencing library. The DNA concentration was measured on a Qubit 3 Fluoromter (ThermoFisher, Cat#Q33216) using Qubit dsDNA HS reagent (ThermoFisher, Cat#Q32851). When there are multiple sample PacBio sequencing libraries, equal amounts of the libraries can be mixed together to prepare a mixed library.

[0076] Step 3: PacBio sequencing and analysis According to the total concentration and molar concentration of the library, an appropriate volume of the library was reacted with binding reagent (PacBio, Cat#101-820-200) and primer (PacBio, Cat#100-970-100) to prepare the final library that can be used in the machine. Representative sequencing results are shown in Figures 3 - 6 As shown, Figure 3 It is a normal sample (SMN1-two copies / SMN2-two copies), Figure 4 The sample is an SMA carrier (SMN1-one copy / SMN2-two copies), Figure 5 It is a sample from an SMA patient (SMN1-zero copy / SMN2-two copies), Figure 6 This is a sample with SMN1 point mutation.

[0077] Embodiment 3: SMN1 / SMN2 Detection and verification of gene mutations The peripheral blood genomic DNA of 25 subjects was collected as validation samples, a total of 25 cases. Referring to Example 2, the method (and kit) of the present invention was used to simultaneously detect the copy number and point mutation of SMN-related genes. At the same time, the copy number was detected by qPCR, and the point mutation and deletion of related genes were detected by Sanger sequencing. The results obtained by the present invention were compared with the control results, and the results are shown in Table 8, among which the results of 25 samples were completely consistent.

[0078]

[0079] Therefore, the results of the detection by the method of the present invention, after comparison with the SMN1 qPCR and Sanger sequencing methods, both the specificity and sensitivity reached 100%.

[0080] It should be noted that although some features of the present invention have been illustrated by the above embodiments, they cannot be used to limit the present invention. For those skilled in the art, the present invention can be modified and varied in various ways. The reaction reagents, reaction conditions, etc. involved in the long-fragment PCR reaction and the construction of the single-molecule sequencing library can be adjusted and changed accordingly according to specific needs. Therefore, for those skilled in the art, several simple substitutions can be made without departing from the concept and principle of the present invention, which should all be included in the scope of protection of the present invention.

[0081] References 1. Beijing Medical Association Medical Genetics Branch, Beijing Rare Disease Diagnosis and Treatment and Protection Society. Expert consensus on genetic diagnosis of spinal muscular atrophy[J]. Chinese Medical Journal, 2020, 100(40): 3130-3140. 2. Mercuri E, Pera MC, Scoto M, Finkel R, Muntoni F. Spinal muscularatrophy — insights and challenges in the treatment era. Nature ReviewsNeurology. 2020;16(12):706-15. 3.Farrar M, Park S, Vucic S, Carey K, Turner B, Gillingwater T, etal. Emerging Therapies and Challenges in Spinal Muscular Atrophy. Annals ofNeurology. 2016;81. 4.Chaytow H, Huang YT, Gillingwater TH, Faller KME. The role of survival motor neuron protein (SMN) in protein homeostasis. Cellular and molecular life sciences: CMLS. 2018;75(21):3877-94. 5.Hatanaka F, Suzuki K, Shojima K, Yu J, Takahashi Y, Sakamoto A, etal. Therapeutic strategy for spinal muscular atrophy by combining genesupplementation and genome editing. Nature Communications. 2024;15(1):6191. 6.Clermont O, Burlet P, Benit P, Chanterau D, Saugier-Veber P,Munnich A, et al. Molecular analysis of SMA patients without homozygous SMN1deletions using a new strategy for identification of SMN1 subtle mutations.Human mutation. 2004;24(5):417-27. 7.Arkblad EL, Darin N, Berg K, Kimber E, Brandberg G, Lindberg C, etal. Multiplex ligation-dependent probe amplification improves diagnostics inspinal muscular atrophy. Neuromuscular disorders : NMD. 2006;16(12):830-8. 8.The next generation of population-based spinal muscular atrophycarrier screening: comprehensive pan-ethnic SMN1 copy-number and sequencevariant analysis by massively parallel sequencing. Genetics in medicine :official journal of the American College of Medical Genetics. 2017;19(8):936-44. 9.eng J, Cui D, Li C, Yang Y, Li Q, Li X, Tan S, Li Z, Meng W, Li H,Zhang Y. The comprehensive analysis of thalassemia alleles (CATSA) based onsingle-molecule real-time technology (SMRT) is a more powerful strategy inthe diagnosis of thalassemia caused by rare variants. Clin Chim Acta. 2023Nov 1;551:117619. 10.Li S, Han X, Zhang L, Xu Y, Chang C, Gao L, et al. An Effectiveand Universal Long-Read Sequencing-Based Approach for SMN1 2 + 0 CarrierScreening through Family Trio Analysis. Clinical chemistry. 2023;69(11):1295-306.

Claims

1. A primer set for simultaneously detecting multiple mutations of SMA pathogenic genes, comprising a SMA-1F / 1R primer pair, a SMA-2F / 2R primer pair and an internal reference gene primer pair; wherein the internal reference gene primer pair is selected from a B2M-F / R primer pair, a GAPDH-F / R primer pair, an ACTB-F / R primer pair or a SDHA-F / R primer pair; The nucleotide sequence of the SMA-1F primer is shown in any one of SEQ ID NOs: 1-3, and the nucleotide sequence of the SMA-1R primer is shown in any one of SEQ ID NOs: 4-6; The nucleotide sequence of the SMA-2F primer is shown in any one of SEQ ID NOs: 7-9, and the nucleotide sequence of the SMA-2R primer is shown in any one of SEQ ID NOs: 10-12; The nucleotide sequence of the B2M-F primer is shown in any one of SEQ ID NOs: 13-15, and the nucleotide sequence of the B2M-R primer is shown in any one of SEQ ID NOs: 16-18; wherein the nucleotide sequence of the GAPDH-F primer is as shown in any one of SEQ ID NOs: 19-21, and wherein the nucleotide sequence of the GAPDH-R primer is as shown in any one of SEQ ID NOs: 22-24; wherein the nucleotide sequence of the ACTB-F primer is shown in SEQ ID NO: 25 or 26, wherein the nucleotide sequence of the ACTB-R primer is shown in SEQ ID NO: 27 or 28; The nucleotide sequence of the SDHA-F primer is shown in SEQ ID NO: 29 or 30, and the nucleotide sequence of the SDHA-R primer is shown in SEQ ID NO: 31 or 32.

2. The primer set according to claim 1, which is used to simultaneously detect SMN1 Genes and SMN2 Copy number variation and point mutations of genes.

3. The primer set according to claim 2, wherein the detection of copy number variation is included in SMN1 Genes and SMN2 The copy number was detected when the gene had only one haplotype and the read ratio was 1:

1.

4. The primer set according to claim 2, wherein the point mutations comprise at least 168 point mutations as shown in Table 1. 5 . The primer set according to claim 1 , wherein the amplification product of the SMA-1F / 1R primer pair, the SMA-2F / 2R primer pair and the internal reference gene primer pair is 15 kb. The primer set according to claim 1 , wherein the 5′ end of the primers in the primer set further comprises a DNA barcode.

7. The primer set according to claim 6, wherein the DNA barcodes are the same or different, and the length of the DNA barcodes is 5-50 nt.

8. The primer set according to claim 1, wherein the primer set is used to simultaneously amplify SMN1 Gene, SMN2 genes and internal reference genes.

9. A kit for simultaneously detecting multiple mutations of SMA pathogenic genes, comprising the following reagents: (1) Reagents for multiplex PCR amplification; (2) Internal reference gene standards for quantitative analysis; (3) Reagents for constructing single-molecule sequencing libraries; The reagents for multiplex PCR amplification comprise the primer set according to any one of claims 1 to 8.

10. The kit according to claim 9, which is used for simultaneous detection SMN1 Genes and SMN2 Copy number variation and point mutations of genes.

11. The kit according to claim 10, wherein the detection of copy number variation comprises SMN1 Genes and SMN2 The copy number was detected when the gene had only one haplotype and the read ratio was 1:

1.

12. The kit according to claim 10, wherein the point mutations comprise at least 168 point mutations as shown in Table 1.

13. The kit according to claim 9, wherein the reagents for multiplex PCR amplification further comprise a DNA polymerase and a reaction buffer.

14. The kit according to claim 9 or 13, wherein the reagents for multiplex PCR amplification are used to simultaneously amplify in one reaction system SMN1 Gene, SMN2 genes and internal reference genes.

15. The kit according to claim 9, wherein the reagents for constructing a single-molecule sequencing library comprise adapters, ligase, DNA purification magnetic beads, reaction buffer and exonuclease.

16. A system for simultaneously detecting multiple mutations of SMA pathogenic genes, comprising the following modules: (1) Collection module: used to obtain and prepare subject samples; (2) Amplification module: used for performing multiplex PCR amplification on the sample; (3) Library construction module: used to construct single-molecule sequencing libraries; (4) Sequencing module: used for sequencing and analyzing mutation types; The multiplex PCR amplification in the acquisition module is performed using a primer set as described in any one of claims 1 to 8 or a kit as described in any one of claims 9 to 15.

17. The system according to claim 16, wherein the multiple mutations of the SMA pathogenic gene comprise SMN1 Genes and SMN2 Copy number variation and point mutations of genes.

18. The system of claim 16, wherein the detection of copy number variation comprises SMN1 Genes and SMN2 The copy number was detected when the gene had only one haplotype and the read ratio was 1:

1.

19. The system according to claim 16, wherein the point mutations comprise at least 168 point mutations as shown in Table 1.

20. The system of claim 16, wherein the sample is selected from a biological sample or gDNA extracted from the biological sample.

21. The system of claim 20, wherein the biological sample comprises cultured cell lines, blood, amniotic fluid, chorionic villi, gametes, blastocyst cells, joint fluid, urine, sweat, saliva, feces, cerebrospinal fluid, ascites, pleural effusion, bile, and pancreatic fluid.

22. The system according to claim 16, wherein the sequencing module is selected from PacBio sequencing of Pacific Biosciences or Nanopore sequencing of ONT.

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