CN115976182A

CN115976182A - Primer probe set and kit for detecting spinal muscular atrophy pathogenic gene SMN1

Info

Publication number: CN115976182A
Application number: CN202210894697.6A
Authority: CN
Inventors: 杨尧; 张鲁军; 郭翠; 韦淑杯; 张伟; 戴国华; 王庆; 张燕娜
Original assignee: Suzhou Ganjiang Biotechnology Co ltd
Current assignee: Suzhou Ganjiang Biotechnology Co ltd
Priority date: 2022-07-28
Filing date: 2022-07-28
Publication date: 2023-04-18

Abstract

The invention discloses a primer probe set and a kit for detecting spinal muscular atrophy pathogenic gene SMN 1. The kit provided by the invention comprises 3 gene detection solutions, wherein the kit is provided with a plurality of primers and probes, and the primers and the probes are respectively shown as SEQ ID NO:1 to SEQ ID NO: shown at 33. The kit provided by the invention overcomes the defects and defects of the prior art, adopts a fluorescence probe melting curve technology to simultaneously detect the copy number of the SMN1 gene and common point mutation, can detect trace samples including dry blood spots/saliva, can simultaneously detect SMA patients and carriers, and has the advantages of short time consumption, simple and convenient operation, strong specificity, high sensitivity, high accuracy and low cost.

Description

Primer probe set and kit for detecting spinal muscular atrophy pathogenic gene SMN1

Technical Field

The invention belongs to the technical field of biology, and relates to a primer probe set and a kit for detecting a spinal muscular atrophy pathogenic gene SMN 1.

Background

Spinal Muscular Atrophy (SMA) is the most common neuromuscular disease in children, and is clinically characterized by muscle weakness and muscle atrophy caused by degeneration of alpha-motor neurons at the anterior angle of the spinal cord. The clinical symptoms of SMA patients vary greatly and can develop diseases from postnatal to adult. It is mainly manifested as progressive muscle weakness and muscular atrophy mainly involving the proximal extremities, and as the disease progresses, there may be multiple system involvement such as respiration, digestion, skeleton, etc.

SMA belongs to autosomal recessive hereditary diseases, and is caused by mutation of a survival gene 1 (survivor neuron 1, SMN 1) of motor neurons, and SMN1 mutation causes degeneration of alpha motor neurons of anterior keratinocytes of spinal cord, which in turn causes severe progressive muscle weakness and muscle weakness, thereby causing early death of patients. The SMN gene has two highly homologous SMN1 and SMN2 genes, and the SMN1 and SMN2 genes are only found to have a difference of 5 bases in the region from exon 6 to exon8 (c.835-44, c.840, INS7+100, INS7+215, c. + 239), and c.840 on exon7, the homology is more than 99 percent, and all encode SMN protein. The SMN1 gene encodes a functional full-length SMN protein, and SMN2 expresses only a small amount of a normally functional protein. SMN1 is a pathogenic gene of SMA, and SMN2 is related to the severity of the disease as a modification compensation gene.

SMA mutant genotypes are mainly of two types, 95% resulting from a biallelic homozygous deletion of the SMN1 gene; 5% are caused by complex heterozygous mutations in the SMN1 gene, i.e., one allele is deleted and the other allele undergoes minor pathogenic variations (including point mutations). It is rare that both SMN1 alleles are slightly pathogenic variants. Most of SMN1 gene deletion is exon7 combined exon8 common deletion, and a few are exon7 deletion. The SMN1 gene micro-pathogenic variation shows different variation pedigrees in different ethnic patients, about 90 types of micro-pathogenic variation (http:// www.hgmd.cf.ac.uk /) have been reported at home and abroad at present, nearly 30 types of micro-pathogenic variation have been reported by Chinese patients, wherein the detection frequency is more than or equal to 2, and 7 types of micro-pathogenic variation are confirmed by the pathogenicity evaluation of the American society for medicine and genetics (ACMG). The most common variations among the chinese population are exon 1 c.22dupa (p.ser8lysfs 23) and exon 5 c.683t > a (p.leu228), accounting for approximately 31.7% and 17.1% of all point mutations, respectively.

The population carrying frequency of SMA is about 1/40-50, the estimated incidence rate is 1/10000-12000 live born, and the SMA is a common autosomal recessive genetic disease. The high disability and lethality of SMA bring heavy influence and burden to the family and the society of patients. The current sequential marketing of disease modifying therapies and gene therapy drugs has brought great hopes to patients. There is increasing evidence that the earlier presymptomatic gene therapy is performed on children patients, the most benefitting is seen in most patients, even though the motor function is normally developed. Early detection by neonatal screening is critical to ensure that effective treatment is achieved before disease symptoms appear. Prenatal cases determined by carrier screening will also allow early treatment. Carrier and neonatal screening of SMA has been routinely conducted in some countries and regions, and the preventive window for SMA has been further advanced.

In 2020, consensus of genetics diagnosticians of spinal muscular atrophy (hereinafter, consensus of genetics diagnosticians) defines that the pathogenic variation of double alleles of SMN1 is the main basis of SMA diagnosis, and clinical diagnosis or clinical suspected SMA patients should be subjected to gene detection. The target genes for gene detection are SMN1 gene and SMN2 gene. Wherein the detection result of the SMN1 gene copy number and the pathogenicity variation is used for disease diagnosis or exclusion diagnosis, and the detection result of the SMN2 gene copy number is used as a reference index for treatment, clinical management and prognosis evaluation after patient diagnosis. Because of the high homology of the SMN1 gene and the SMN2 gene in the human genome, gene detection needs to be performed on the SMN1 gene and the SMN2 gene respectively. The full-length transcripts of SMN1 and SMN2 genes were at base-differential sites in exon7 (c.840C/T) and exon8 (c.about.239G/A) as the primary reference sites for distinguishing the two loci. Since 95% of SMA patients are homozygous deletions of exon7 of the SMN1 gene, quantitative analysis of the copy number of exon7 of the SMN1 gene should be performed first. If the 7 th exon homozygous deletion or the 7 th and 8 th exons homozygous deletion of the SMN1 gene is detected, the SMN1 homozygous deletion type patient can be diagnosed. For non-homozygous deletion variants, minor pathogenic mutations in the SMN1 gene must be analyzed and confirmed. In patients with well-defined genetic diagnosis, the parents must perform SMN1 genetic testing to determine the origin of the parent SMN1 genotype and patient variant genes and to perform genetic counseling.

Commonly used methods for detecting the copy number of the SMN1 gene include Multiplex Ligation Probe Amplification (MLPA), quantitative polymerase chain reaction (qPCR). The traditional detection method of SMA also comprises 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 and the like. MLPA can definitely distinguish patients, carriers or normal persons by directly detecting the copy number of SMN1, and can also detect the copy number of SMN2 at the same time, but the MLPA has complex operation and high cost, needs anticoagulation extraction of purified genomic DNA, and is not suitable for large-scale newborn screening. The qPCR uses housekeeping gene sequence as internal reference, and adopts Taqman probe method to respectively carry out relative quantification on SMN 17 th or 8 th exon copy number so as to judge whether deletion variation occurs; the method is simple to operate and low in cost, anticoagulant extraction of purified genome DNA is also needed, and direct extraction and detection of dry blood spots can cause the amplification efficiency of a target gene and an internal reference gene to be different between samples, so that false positive and false negative results are easy to occur in detection of carriers.

In the prior art, a melting curve method by adding a fluorescent dye or a fluorescent probe is adopted to detect the copy number of the SMN1 gene, but probes aiming at a target region and an internal reference region are respectively designed, melting curve analysis is carried out by amplifying the two regions, the copy number variation of the target region is detected by the melting peak intensity ratio of the two regions, the target region and the internal reference region have different gene sequences, the gene amplification efficiency has difference, in order to eliminate the influence of different amplification efficiencies, the limited amplification is carried out by adopting extremely low dNTP concentration, but the quantity of amplified products is obviously reduced, the detection sensitivity is influenced, and the requirement for detecting a trace sample of dried blood spots of a newborn is difficult to meet. The direct design of fluorescent probes for detection of SMN1 gene and SMN2 gene at exon7 different base sites (c.840C/T) can only detect patients with double-copy complete deletion of SMN1, and cannot detect carriers with single-copy deletion, because SMN1 and SMN2 copy number 1 cannot be distinguished: 1 and 2: 2.

The conventional method mainly uses an anticoagulant sample to detect the copy number of the SMN1 exon7 as a main part, and cannot detect small variation of the SMN1 gene. Therefore, the screening may have omission, and some researches show that the high-throughput sequencing technology can simultaneously detect the copy number of the SMN1 gene and screen the small variation of the SMN gene, but the high-throughput sequencing technology has complex operation, complex steps and high instrument and reagent cost, and is not suitable for large-scale newborn screening.

Disclosure of Invention

The invention aims to provide a primer probe set and a kit for detecting a spinal muscular atrophy pathogenic gene SMN 1. The kit provided by the invention overcomes the defects and defects of the prior art, simultaneously detects the SMN1 gene copy number and common point mutation by adopting a fluorescent probe melting curve technology, can detect trace samples including dry blood spots/saliva, can simultaneously detect SMA patients and carriers, and has the advantages of short time consumption, simple and convenient operation, strong specificity, high sensitivity, high accuracy and low cost.

The SMN1 gene is collectively called a motor neuron survival gene 1 (survivor neuron gene 1).

The invention provides a kit, which comprises a gene detection solution A, a gene detection solution B and a gene detection solution C.

The kit also comprises a pretreatment solution and an amplification reaction solution.

The invention also protects the application of the gene detection solution A, the gene detection solution B and the gene detection solution C in the preparation of the kit.

The invention also protects the application of the gene detection solution A, the gene detection solution B, the gene detection solution C, the pretreatment solution and the amplification reaction solution in the preparation of the kit.

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

The gene detection solution B contains a primer F4, a primer R4, a primer F6, a primer R6, a primer F5, a primer R5, a probe FP6, a probe Q4, a probe FP7, a probe FP11, a probe Q7, a probe FP5 and a probe Q3.

The gene detection solution C contains a primer F1, a primer R1, a primer F2, a primer R2, a primer F3, a primer R3, a probe FP1, a probe FP2, a probe Q1, a probe FP3 and a probe FB1.

In the gene detection solution A, the molar ratio of a primer F4, a primer R4, a primer F6, a primer R6, a primer F7, a primer R7, a probe FP4, a probe Q2, a probe FP8, a probe Q5, a probe FP9, a probe FP10 and a probe Q6 is as follows in sequence: 1.5:15:12:1.2:1:10:6:6:6:6:5:5:5.

in the gene detection solution B, the molar ratio of a primer F4, a primer R4, a primer F6, a primer R6, a primer F5, a primer R5, a probe FP6, a probe Q4, a probe FP7, a probe FP11, a probe Q7, a probe FP5 and a probe Q3 is as follows in sequence: 1.5:15:1:10:1.5:15:7.5:7.5:7.5:5:5:6:6.

in the gene detection solution C, the molar ratio of the primer F1, the primer R1, the primer F2, the primer R2, the primer F3, the primer R3, the probe FP1, the probe FP2, the probe Q1, the probe FP3 and the probe FB1 is as follows in sequence: 1:10:1:10:10:1:5:5:5:5:2.

the pretreatment liquid contains Chelex-100, triton-X100 and NaOH.

Specifically, the pretreatment liquid: comprises 5 percent (mass percent) of Chelex-100, 0.2 percent (volume percent) of Triton-X100, 40mM NaOH and the balance of water.

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

Specifically, the composition of the amplification reaction solution was shown in Table 4 for 1 person (6.0. Mu.l).

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

The invention also protects the application of the primer probe group in the preparation of a kit.

The invention also provides a kit comprising the primer probe group.

The function of any one of the kits is as follows (a) or (b): (a) detecting SMN1 gene mutation; (b) screening spinal muscular atrophy patients; (c) screening spinal muscular atrophy carriers.

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

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

The probe FP4, the probe FP8, the probe FP9 and the probe FP10 are labeled with different fluorophores (i.e., 4 probes have four fluorophores).

Probe FP6, probe FP7, probe FP11 and probe FP5 are labeled with different fluorophores (i.e., 4 probes have four fluorophores).

The probe FP1, the probe FP2 and the probe FP3 are labeled with different fluorophores (i.e., 4 probes have three fluorophores).

The end of probe FP1,5 'is marked with a fluorescent group, and the end of 3' is marked with a fluorescence quenching group;

labeling a fluorescent group at the end of the probe FP2,5', and modifying the end of the probe FP 3' by using a C3 Spacer;

the tail ends of the probes Q1 and Q3' are marked with fluorescence quenching groups;

the 3,5 'end of the probe FP is marked with a fluorescent group, and the 3' end is marked with a fluorescence quenching group;

labeling a fluorescent group at the end of 4,5 'of the probe FP, and modifying the end of 3' by using a C3 Spacer;

the tail end of the probe Q2,3' is marked with a fluorescence quenching group;

labeling a fluorescent group at the 5,5 'end of the probe FP, and modifying the 3' end by using a C3 Spacer;

the tail end of the probe Q3,3' is marked with a fluorescence quenching group;

labeling a fluorescent group at the 3' end of the probe FP 6;

the 5 'end of the probe Q4 is marked with a fluorescence quenching group, and the 3' end is marked with a fluorescence quenching group;

labeling a fluorescent group at the 5 'end of the probe FP7, and modifying the 3' end by using a C3 Spacer;

labeling a fluorescent group at the 5 'end of the probe FP8, and modifying the 3' end by using a C3 Spacer;

the tail end of the probe Q5,3' is marked with a fluorescence quenching group;

labeling a fluorescent group at the end of the probe FP9, 3';

the 5 'end of the probe Q6 is marked with a fluorescence quenching group, and the 3' end is marked with a fluorescence quenching group;

labeling a fluorescent group at the 5 'end of the probe FP10, and modifying the 3' end by using a C3 Spacer;

labeling a fluorescent group at the 5 'end of the probe FP11, and modifying a C3Spacer at the 3' end;

the probe Q7,3' end is marked with a fluorescence quenching group.

The end of probe FP1,5 'is labeled with FAM, and the end of 3' is labeled with BHQ2;

marking CY5 and 3 'ends at the 2,5' ends of the probe FP for C3Spacer modification;

the end of the probe Q1,3' is marked with BHQ2;

probe FP3,5 'end mark ROX,3' end mark BHQ2;

carrying out locked nucleic acid modification on 12 th nucleotide and 13 th nucleotide of the probe FB1, and carrying out C3Spacer modification at the 3' end;

marking CY5 at the end of probe FP4,5 'and carrying out C3Spacer modification at the end of 3';

the BHQ2 is marked at the tail end of the probe Q2 and 3';

ROX is marked at the 5,5 'end of the probe FP, and C3Spacer modification is carried out at the 3' end;

BHQ2 is marked at the tail end of the probe Q3 and 3';

labeling FAM at the 3' end of the probe FP 6;

the 5 'end of the probe Q4 is marked with BHQ1, and the 3' end is marked with BHQ1;

HEX is marked at the 5 'end of the probe FP7, and C3Spacer modification is carried out at the 3' end;

ROX is marked at the 5 'end of the probe FP8, and C3Spacer modification is carried out at the 3' end;

the MGB is marked at the 5,3' end of the probe Q;

labeling FAM at the 3' end of the probe FP 9;

the 5 'end of the probe Q6 is marked with BHQ1, and the 3' end is marked with BHQ1;

HEX is marked at the end of 10,5 'of the probe FP, and C3Spacer modification is carried out at the end of 3';

marking CY5 at the 5 'end of the probe FP11, and carrying out C3Spacer modification at the 3' end;

the 3' end of probe Q7 is labeled with MGB.

The working principle of the kit provided by the invention is principle one, principle two and principle three.

Schematic diagrams of principle one and principle two are shown in fig. 1.

Principle one is as follows:

designing an upstream primer and a downstream primer aiming at two targets of exon7 (c.840C/T; C.840C for SMN1 gene, C.840T for SMN2 gene) and exon8 (c.a. 239G/A; C.a. 239G for SMN1 gene and c.a. 239A for SMN2 gene), and amplifying single-stranded DNA containing a target region by adopting asymmetric PCR.

And designing a fluorescence detection probe according to the target spot, wherein the fluorescence detection probe is a self-quenching probe or an adjacent quenching probe group. One end of the self-quenching probe is marked with a fluorescent group, and the other end of the self-quenching probe is marked with a fluorescent quenching group; when no target exists, the probe is naturally curled, and the fluorescent group is close to the fluorescence quenching group, so that the fluorescence is quenched; when the target exists, the probe is hybridized with the target, the distance between the fluorescent group and the fluorescence quenching group is pulled apart, and thus a fluorescent signal appears. The adjacent quenching probe set consists of two probes, wherein one probe is a fluorescent probe (marked with a fluorescent group, and a target region of the fluorescent probe covers a target site), and the other probe is a quenching probe (marked with a fluorescent quenching group, and a target region of the fluorescent quenching probe is adjacent to a target region of the fluorescent probe); when the target exists, the fluorescent probe and the quenching probe are hybridized on the target, the distance is close, and the fluorescent group is quenched by the fluorescence quenching group; when the target does not exist, the distance between the fluorescent probe and the quenching probe is pulled apart, and the fluorescent probe generates a fluorescent signal.

And c.840 sites and c.239 sites have different typing and probe binding forces, so that Tm in a melting curve is different, and the genotyping is judged according to the distribution of Tm values. By using the difference site between the SMN1 gene and the SMN2 gene, the probe is completely matched with the SMN1 gene and is not completely matched with the SMN2 gene, so that the amplification product of the SMN1 gene generates a melting peak with a high Tm value, and the amplification product of the SMN2 gene generates a melting peak with a low Tm value. If the melting peak is missing at a high Tm, it indicates that both copies of the SMN1 gene are completely missing. If the high Tm value melting peak and the low Tm value melting peak are equal in intensity, it is likely that the expression level of the "SMN1 gene: SMN2 gene copy number 1:1", possibly also the" SMN1 gene: SMN2 gene copy number 2:2", it cannot be directly judged whether or not the SMN1 gene is deleted in a single copy.

The second principle is as follows:

in order to realize the analysis of single copy deletion (carrier) of the SMN1 gene, a homologous gene amplification comparative analysis method is adopted, a Target region (SMN 1-Target) is selected near an exon7 region of the SMN1 gene, a reference region (SMN 1-Ref) highly homologous to the Target region is compared in the whole genome, the number of highly homologous regions is not more than 2, PCR amplification upstream and downstream primers are designed aiming at the screened highly homologous regions, and the SMN1-Target and the SMN1-Ref are simultaneously amplified by adopting the same primers. Because the SMN1 gene and the SMN2 gene are highly homologous, in order to avoid the interference of the SMN2 gene, a blocking primer (the 3 'end of the primer is 1 base difference from the SMN1 gene and 2 base difference from the SMN2 gene) is designed according to the difference site INS7+100 (SMN 1 is INS7+100A, SMN2 is INS7+ 100G) of the SMN1 gene and the SMN2 gene, and a blocking probe (the blocking probe covers the INS7+100 site and the 3' end position of the primer, the blocking probe is completely matched with the INS7+100G of the SMN2 gene and is modified by a locked nucleotide at the INS7+100G base to ensure that the blocking probe is preferentially combined with the SMN2 gene to avoid the interference of the SMN2 gene) is designed. And designing a fluorescent detection probe, wherein a hybridization region of the fluorescent detection probe selects sequences with base difference in SMN1-Target and SMN1-Ref, so that the SMN1-Target and SMN1-Ref are distinguished through melting peaks with different Tm values, the melting peak with a high Tm value is an SMN1 gene amplification product, and the melting peak with a low Tm value is a genome homologous reference region amplification product. When the SMN1 gene is subjected to copy number deletion, the template ratio of the SMN1 gene to a homologous reference region is changed from 2:2 to 1:2 or 0:2, because the same amplification primer is adopted, when the template ratio is obviously changed, the ratio of amplification products is also obviously changed, so that the intensity ratio of melting peaks is changed, and the detection of single copy deletion and double copy deletion of the SMN1 gene is carried out.

The principle three is as follows:

in order to realize the simultaneous analysis of common mutation sites of the SMN1 gene, an amplification primer is designed aiming at the most common mutation sites (see table 1) in Chinese population. Primers are designed to amplify the corresponding mutation sites respectively. And designing a fluorescence detection probe according to the target spot, wherein the fluorescence detection probe is a self-quenching probe or an adjacent quenching probe group. If the detection site is mutated, the melting peak of the probe is changed, and a melting peak with a low Tm value or a melting peak with a high Tm value is generated, thereby judging whether the mutant is a wild type, a heterozygous mutant or a homozygous mutant.

TABLE 1

Serial number	Mutant sites and mutant forms
		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)

The kit provided by the invention can detect double-copy deletion, single-copy carrying and common mutation (namely mutation shown in table 1) of the SMN1 gene, thereby reducing the omission factor. The kit provided by the invention adopts homologous region amplification, uses normal dNTP concentration, specifically detects copy number variation of a target region, and does not influence detection sensitivity. The gene detection reagent in the prior art needs more blood samples and purifies genome DNA, and is not suitable for screening dry blood spots of newborn infants. The kit provided by the invention can be used for directly pretreating a trace blood sample (3 mu l of blood or 3mm dried blood spots), and the treatment solution is directly used for subsequent amplification detection without complicated steps such as genome DNA purification and the like, so that the cost and the time are saved, and the kit is suitable for screening and detecting the dried blood spots of the newborn. The kit provided by the invention can be used for carrying out SMN1 gene carrier detection on trace saliva samples, is suitable for carrying out carrier screening in normal people and pregnant woman, and effectively carries out three-level prevention and control on SMA.

The invention has the beneficial effects that: (1) The SMN1 gene detection is completed through three PCR reactions, and the homozygous deletion of exons 7 and 8, the single copy deletion of the exon7 and common mutation (namely the mutation shown in the table 1) are detected at the same time, so that the missed detection can be effectively reduced, the detection cost is low, the flux is high, the operation is convenient and fast, and the time consumption is short (the detection is completed within 2.5 hours); (2) the sensitivity reaches 2ng; (3) The direct detection of trace dry blood spots can be realized, and the newborn screening is facilitated; (4) The direct detection of a trace saliva sample can be realized, and the noninvasive detection method is favorable for screening carriers of large-scale people in a noninvasive mode; (5) A fluorescence probe multiple melting curve is adopted, homologous gene comparison analysis is adopted, an amplification primer and a probe in a region near an SMN1 exon7 are designed according to a genome homologous sequence, CNV detection is realized, and the detection specificity is improved compared with a Taqman probe method.

Drawings

FIG. 1 shows the principle of amplification of exon7 and exon8 regions of the SMN1 gene.

FIG. 2 shows the effect of inhibiting SMN2 amplification.

FIG. 3 is a comparison of SMN1 gene point mutation detection probe system.

FIG. 4 shows a comparison of SMN1 gene exon7 detection probe system.

FIG. 5 shows a comparison of SMN1 gene exon8 detection probe systems.

FIG. 6 shows a comparison of SMN1 gene copy number detection probe systems.

FIG. 7 shows SMN1 gene point mutation detection plasmids.

FIG. 8 shows SMN1 gene point mutation detection plasmids.

FIG. 9 is a graph of the effect of clinical SMA patient testing.

FIG. 10 is a graph of the effect of clinical SMA carrier testing.

FIG. 11 is a graph showing the effect of clinical normal sample detection.

FIG. 12 shows different copy number measurements of SMN1 in clinical samples.

FIG. 13 shows the detection of SMN1 gene c.22dupA heterozygous mutation.

FIG. 14 shows the detection of SMN1 gene c.400G > A heterozygous mutation.

FIG. 15 shows the detection of SMN1 gene c.863G > T heterozygous mutation.

FIG. 16 shows the effect of different copy numbers of SMN2 on SMN1 detection.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way. The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Unless otherwise stated, the quantitative tests in the following examples were performed in triplicate, and the results were averaged.

Example 1 preparation and use of SMN1 Gene detection kit

1. Design, screening and preparation of primers

Primers are designed aiming at mutation site regions in exons 7 and 8 of SMN1 and SMN2 genes, and the primers with the best performance (specificity and sensitivity) are obtained by combining and screening through a preliminary experiment. Primers designed according to principle one were as follows: an upstream primer F1 and a downstream primer R1, which are used for amplifying exon7 regions (containing c.840 sites) of SMN1 genes and SMN2 genes; and the upstream primer F2 and the downstream primer R2 are used for amplifying exon8 regions (containing c.multidot.239 sites) of the SMN1 gene and the SMN2 gene.

And selecting a sequence from the exon7 region of the SMN1 gene to the IVS7+100 site, and searching the homologous regions in other regions on the whole genome to find that one homologous region exists. Sequences between IVS7+ 21-IVS 7+126 of the SMN1 gene are selected as detection Target sequences SMN1-Target, and the chromosome position is Chr5:70951950-70952192; the homologous sequence on the genome is located in Chr9:20332239-20332475 and is selected as reference sequence SMN1-Ref. According to the homologous sequence alignment information, a primer region is selected from the upstream primer F3 as an amplification upstream primer region and cgc is added at the 5' end to increase the Tm value of the primer, and a blocking primer principle is adopted in the downstream primer R3 to mutate a base T close to the 3' end into c and increase cgg at the 5' end to increase the Tm value of the primer. Primers designed according to principle two were as follows: an upstream primer F3 and a downstream primer R3, which are used for simultaneously amplifying the SMN1 target sequence region and the homologous reference sequence region on chromosome 9.

And designing a primer based on sequence analysis, comparing the effect of the primer through a pre-experiment, and screening to obtain the optimal primer. Primers designed according to principle three were as follows: an upstream primer F4 and a downstream primer R4 are used for amplifying a region comprising 5 variation sites (c.22dupA, c.5C > G, c.40G > T, c.43C > T and c.56delT) in the SMN1 gene; an upstream primer F5 and a downstream primer R5 which are used for amplifying a region comprising 2 mutation sites (c.683T > A and c.689C > T) in the SMN1 gene; an upstream primer F6 and a downstream primer R6 are used for amplifying a region comprising 2 mutation sites (c.863G > T, c.835-5T > G) in the SMN1 gene; the upstream primer F7 and the downstream primer R7 are used for amplifying a region comprising 2 variation sites (c.463 _464delAA, c.400G > A) in the SMN1 gene.

The primers were synthesized by Biotechnology engineering (Shanghai) Co., ltd. The dry powder of the primers was dissolved in TE buffer (10 mM Tris-HCl pH8.0,1mM EDTA pH 8.0) and quantified to 20. Mu.M using a microspectrophotometer.

The information about the primers and the nucleotide sequences are shown in Table 2.

TABLE 2 amplification sites or regions and their corresponding primers

2. Preparation of Probe

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

The self-quenching probe is used independently, the 5 'end and the 3' end of the probe are respectively modified with a fluorescent group and a fluorescence quenching group, the fluorescent group is any one of FAM, HEX, CY5 and ROX, and the fluorescence quenching group is any one of BHQ1, BHQ2 and BHQ3 which can be matched with the fluorescent group for use.

The adjacent quenching probe group is matched by combining a fluorescent probe and a quenching probe. The 5' end of the fluorescent probe is marked with a fluorescent group, the 3' end of the fluorescent probe is subjected to C3Spacer modification treatment, and the 3' end of the quenching probe matched with the fluorescent probe is marked with a fluorescent quenching group. Or, the 3 'end of the fluorescent probe is labeled with a fluorescent group, the 5' end of the quenching probe matched with the fluorescent probe is labeled with a fluorescent quenching group, and the 3 'end is modified by C3Spacer (aiming at the quenching probe of a certain fluorescent probe) or the 3' end is labeled with a fluorescent quenching group (aiming at the quenching probes of two fluorescent probes). The fluorescent group is any one of FAM, HEX, CY5 and ROX, and the fluorescence quenching group is any one of BHQ1, BHQ2, BHQ3 or MGB which can be matched with the fluorescent group for use. C3Spacer is a blocking group that prevents probe extension as a primer.

The blocked probe is not modified by a fluorescent group, and the 3' end is modified by a C3 Spacer.

The probe FP1 is a self-quenching probe, covers the c.840 site of the SMN1 gene, has a sequence completely matched with the SMN1 gene and is not matched with the SMN2 gene at the c.840 site, so that the Tm value of the combination of the probe FP1 and an SMN2 amplification sequence is lower than the Tm value of the combination of the probe FP1 and the SMN1 amplification sequence, thereby detecting the exon7 of the SMN1 gene and the SMN2 gene.

And the probe FP2 is a fluorescent probe, the probe Q1 is a quenching probe, the probe Q1 and the quenching probe form an adjacent quenching probe set, the adjacent quenching probe set covers the site c.239 of the SMN1 gene, the sequence is completely matched with the SMN1 gene, and the adjacent quenching probe set is not matched with the SMN2 gene at the site c.239, so that the Tm value of the combination of the probe FP2 and the SMN2 amplification sequence is lower than the Tm value of the combination of the probe FP2 and the SMN1 amplification sequence, and the exon8 detection of the SMN1 gene and the SMN2 gene is carried out.

The probe FP3 is a self-quenching probe, the sequence of the probe FP3 is completely complementary with a Target sequence in SMN1-Target, and the difference of 4 bases exists in a homologous reference sequence in SMN1-Ref, therefore, an amplification product corresponding to the probe FP3 and the SMN1 Target sequence SMN1-Target generates a melting peak with a high Tm value, an amplification product corresponding to the homologous reference sequence SMN1-Ref generates a melting peak with a low Tm value, and the copy number ratio of the SMN1 gene and the homologous reference sequence is detected according to the ratio of the two melting peaks, thereby realizing the detection of single copy deletion and double copy deletion of the SMN1 gene.

The probe FB1 is a closed probe, is designed aiming at IVS7+100 sites of the SMN1/2 gene, is completely matched with the IVS7+100 sites of the SMN2 gene, carries out locked nucleic acid modification at IVS7+100 sites of differential sites, preferentially combines the SMN2 gene, improves the Tm value difference, improves the closing effect, and carries out C3Spacer modification treatment at the 3' end.

The probe FP4 is a fluorescent probe, the probe Q2 is a quenching probe, the fluorescent probe and the quenching probe form an adjacent quenching probe set, the c.22dupA locus of the SMN1 gene is covered, the sequence is completely matched with the wild type of the SMN1 gene, if the detection locus is mutated, the melting peak of the probe is changed, and the melting peak with a low Tm value is generated, so that whether the detection locus is the wild type, the heterozygous mutant type or the homozygous mutant type is judged.

The probe FP5 is a fluorescent probe, the probe Q3 is a quenching probe, the two probes form an adjacent quenching probe group, cover the C.683T > A locus and the C.689C > T locus of the SMN1 gene, and the sequences are completely matched with the C.683T > A wild type and the C.689C > T mutant type of the SMN1 gene, namely the probe sequences are the c.683T and c.689T genotypes; if the detection site c.683T > A has mutation, the melting peak of the probe can be changed, and a melting peak with a low Tm value appears; if the detection site c.689C > T generates mutation, the melting peak of the probe can be changed, and the melting peak with high Tm value appears; thereby judging whether the mutant is a wild type, a heterozygous mutant or a homozygous mutant.

The probe FP6 is a fluorescent probe, the probe Q4 is a quenching probe (the probe Q4 is matched with the probe FP6 and the probe FP7 at the same time), the two probes form an adjacent quenching probe group, the C.5C > G sites of the SMN1 gene are covered, the sequence is completely matched with the wild type of the SMN1 gene, if the detection sites are mutated, the melting peak of the probe is changed, the melting peak with a low Tm value is generated, and the wild type, the heterozygous mutant type or the homozygous mutant type is judged.

The probe FP7 is a fluorescent probe, the probe Q4 is a quenching probe (the probe Q4 is matched with the probe FP6 and the probe FP7 at the same time), the two probes form an adjacent quenching probe group, cover the C.40G > T site, the c.43C > T site and the c.56delT site of the SMN1 gene, and have sequences completely matched with the C.40G > T site mutant type, the c.43C > T site wild type and the c.56delT site wild type of the SMN1 gene, namely the probe sequences are c.40T, c.43C and c.56T genotypes; if the detection site c.40G > T is mutated, the melting peak of the probe is changed, and a melting peak with a high Tm value appears; if the detection site c.43C > T has mutation, the melting peak of the probe can be changed, and the melting peak with a low Tm value appears; if the detection site c.56delT is mutated, the melting peak of the probe is changed, and the melting peak with a low Tm value appears; thereby judging whether the mutant is a wild type, a heterozygous mutant or a homozygous mutant.

The probe FP8 is a fluorescent probe, the probe Q5 is a quenching probe, the fluorescent probe and the quenching probe form adjacent quenching probe sets, the adjacent quenching probe sets cover C.863G > T sites of the SMN1 gene, the sequences are completely matched with the wild type of the SMN1 gene, if the detection sites are mutated, the melting peak of the probe is changed, and the melting peak with a low Tm value appears, so that whether the wild type, the heterozygous mutant type or the homozygous mutant type is judged.

The probe FP9 is a fluorescent probe, the probe Q6 is a quenching probe (the probe Q6 is matched with the probe FP9 and the probe FP10 at the same time), the probe FP9 and the probe FP10 form an adjacent quenching probe set, the C.400G A site of the SMN1 gene is covered, the sequence is completely matched with the wild type of the SMN1 gene, if the detection site is mutated, the melting peak of the probe is changed, the melting peak with a low Tm value is generated, and therefore whether the detection site is the wild type, the heterozygous mutant or the homozygous mutant is judged.

The probe FP10 is a fluorescent probe, the probe Q6 is a quenching probe (the probe Q6 is matched with the probe FP9 and the probe FP10 at the same time), the two probes form an adjacent quenching probe group, the c.463_464delAA site of the SMN1 gene is covered, the sequence is completely matched with the SMN1 gene mutant type, if the detection site is mutated, the melting peak of the probe is changed, the melting peak with a high Tm value is generated, and the wild type, the heterozygous mutant type or the homozygous mutant type is judged.

And the probe FP11 is a fluorescent probe, the probe Q7 is a quenching probe, the fluorescent probe and the quenching probe form adjacent quenching probe sets, the c.835-5T > -G sites of the SMN1 gene are covered, the sequences of the probes are completely matched with the wild type of the SMN1 gene, if the detection sites are mutated, the melting peak of the probes is changed, and the melting peak with a low Tm value appears, so that whether the probes are the wild type, the heterozygous mutant type or the homozygous mutant type is judged.

The probe is synthesized and modified by Biotechnology engineering (Shanghai) GmbH. The probe dry powder was dissolved in TE buffer (10 mM Tris-HCl pH8.0,1mM EDTA pH 8.0) and quantified to 20. Mu.M using a microspectrophotometer.

The information about the probes and the nucleotide sequences are shown in Table 3.

TABLE 3 information about probes and nucleotide sequences

3. Preparation of SMN1 gene detection kit

The SMN1 gene detection kit (100 persons) consists of the following components: 15ml of pretreatment solution, 2ml of amplification reaction solution, 650. Mu.l of gene detection solution A, 650. Mu.l of gene detection solution B, 650. Mu.l of gene detection solution C, a standard control and 500. Mu.l of negative control solution.

Pretreatment liquid: comprises 5 percent (mass percent) of Chelex-100, 0.2 percent (volume percent) of Triton-X100, 40mM NaOH and the balance of water. Chelex-100 (i.e. the product of the design is a chemical product of the formula)

100 chemical Resin): berle Bio-rad, cat No. 1421253.Triton-X100: biotechnology engineering (Shanghai) Inc., A110694-0100.

The composition of 1-person (6.0. Mu.l) amplification reaction solution is shown in Table 4.

TABLE 4

Reagent	1 person portion (mu l)
		5X Hotstart Taq Buffer	5
Hotstart Taq DNA Polymerase	0.4
		dNTP Mixture	0.5
dUTP	0.05
		2U/. Mu.l UDG enzyme	0.05
In total	6.0

The reagents in Table 4 are all products of Biotechnology (Shanghai) Inc. The product has a product number of B110003-2500, and comprises Hotstart Taq DNA Polymerase (the specification is 5U/. Mu.l) and 5X Hotstart Taq Buffer. dNTP mix (specification 10 mM), cat # B110044-0005.dUTP (specification 100 mM), cat # B110052-0250.UDG enzyme (specification 2U/. Mu.l), cat # B110042-0200.

The composition of the gene assaying liquid A for 1 reaction (total volume 6. Mu.l) is shown in Table 5. The primers and probes in the gene detection solution A are used for detecting c.22dupA, c.863G > T, c.400G > A and c.463_464delAA.

TABLE 5

The composition of the gene assaying liquid B for 1 reaction (total volume 6. Mu.l) is shown in Table 6. The primers and probes in the gene detection solution B were used for detection of c.5C > G, c.40G > T, c.43C > T, c.56delT, c.835-5T >G, c.683T > A, and c.689C > T.

TABLE 6

The composition of the gene assaying liquid C for 1 reaction (total volume 6. Mu.l) is shown in Table 7. The primer and the probe of C in the gene detection solution are used for detecting the exon7 and the exon8 of the SMN1 gene, SMN1-Target and SMN1-Ref.

TABLE 7

Standard control: normal human dry blood spots (both SMN1 and SMN2 genes are wild-type).

Negative control solution: deionized water.

4. Method of using kit

1. Pretreatment

Taking a sample to be detected (the sample to be detected is 3 mu l of whole blood, 10 mu l of saliva or 3mm dry blood spots), adding 100 mu l of pretreatment liquid, incubating for 10min at 95 ℃, centrifuging for 5min at 2500rpm, and collecting supernatant, namely sample template solution.

Taking a standard control, adding 100 mu l of pretreatment solution, incubating for 10min at 95 ℃, then centrifuging for 5min at 2500rpm, and collecting supernatant, namely the standard control solution.

2. Preparation of the detection reaction System

Mixing 6.0 mul of amplification reaction solution, 2 mul of sample template solution and 6 mul of gene detection solution A, adding deionized water to 25 mul, centrifuging to remove bubbles, and obtaining a sample detection reaction system A.

And mixing 6.0 mul of amplification reaction solution, 2 mul of sample template solution and 6 mul of gene detection solution B, adding deionized water to 25 mul, and centrifuging to remove bubbles to obtain a sample detection reaction system B.

Mixing 6.0 mul of amplification reaction solution, 2 mul of sample template solution and 6 mul of gene detection solution C, adding deionized water to 25 mul, centrifuging to remove bubbles, and obtaining a sample detection reaction system C.

And replacing the sample template solution with the standard control solution to respectively obtain three standard control detection reaction systems.

And replacing the sample template solution with the negative control solution to respectively obtain three standard control detection reaction systems.

3. Detection of

And (3) carrying out PCR amplification and melting curve analysis on the detection reaction system by adopting a fluorescent quantitative PCR instrument (Shanghai Hongshi SLAN-96S).

PCR amplification procedure: 2min at 50 ℃ and 10min at 95 ℃;95 ℃ 20s, 56 ℃ 60s,45 cycles.

Melting curve analysis program: 1min at 95 ℃ and 3min at 37 ℃; the temperature is increased from 40 ℃ to 85 ℃ at the speed of 0.04 ℃/s, the melting curve analysis is carried out, and the fluorescence signals of all the fluorophores are collected at the same time.

4. Analysis of results

And obtaining the Tm value of each channel of the reaction detection system by the fluorescent quantitative PCR instrument configuration software according to the fluorescent signal. The Tm value is subject to automatic interpretation of the instrument, but when the instrument cannot give the Tm value, the Tm value can be obtained by adjusting a baseline or manually interpreting.

Quality control: for the standard control, each corresponding fluorescence channel in the reaction system needs to have a melting peak with Tm in the reference value range (the reference value range of Tm of the melting peak of the standard control is shown in Table 8); for the negative control solution, no distinct melting peak is required for each corresponding fluorescence channel in the reaction system. If the quality control is satisfied, the detection amplification system is effective.

And for the sample to be detected, a melting peak appears in each fluorescence signal channel in the reaction system, the melting peak is compared with the corresponding melting peak of the standard control, and SNP typing is judged according to the Tm difference. Only the wild type peak is found, the genotype corresponding to the gene locus is the homozygous wild type. If only mutant peak exists, the genotype corresponding to the gene locus is homozygous mutant. If the wild type peak and the mutant type peak appear at the same time, the corresponding gene locus is a heterozygote of the wild type and the mutant type.

TABLE 8 reference value ranges for Tm of melting peaks of standard controls

Reaction System A and reaction System B for examinationThe common mutation sites of the SMN1 gene listed in Table 1 are determined according to the following analysis scheme: the Tm value of each channel of the sample detection reaction system is recorded as Tm, and the Tm value of each channel of the standard control detection reaction system is recorded as Tm _{Standard control} By the formula Δ Tm = Tm-Tm _{Standard control} Obtaining a delta Tm value, and determining whether the corresponding gene mutation exists in the sample to be detected and the mutation type according to the delta Tm value. And judging whether the sample to be detected contains corresponding gene mutation or not and the type of the gene mutation according to the change of the Tm values of the melting peaks of the sample to be detected and the standard control in each channel in the analysis result of the melting curve of the multicolor probe. This involves the reading of the Tm values of the sample to be tested and of the standard control (obtained automatically by the fluorescence PCR instrument configuration software) as a correction to reduce the melting point errors caused by different instruments and human operations. Subtracting the Tm value of each channel of the sample to be detected from the Tm value of each channel of the standard control to obtain the Delta Tm of each channel, and judging whether the sample to be detected contains mutation or not and the type of the mutation according to the Delta Tm value of each channel (tables 9 and 10).

TABLE 9

TABLE 10

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Reaction system C was used to detect copy number variation of exon7 and exon8 of the SMN1 gene.

FAM fluorescence channel: the high Tm melting peak is SMN1-Exon7, and the low Tm melting peak is SMN2-Exon7; when exons 7 of the SMN1 gene and the SMN2 gene of a normal sample exist, signals exist in both a high Tm melting peak and a low Tm melting peak of an FAM channel; if the high Tm melting peak is deleted, the SMN1-Exon7 double copy deletion is shown; if the low Tm melting peak is missing, it indicates that SMN2-Exon7 double copies are missing.

CY5 fluorescence channel: the high Tm melting peak is SMN1-Exon8, and the low Tm melting peak is SMN2-Exon8; when exons 8 of the SMN1 gene and the SMN2 gene of a normal sample exist, signals exist in both a high Tm melting peak and a low Tm melting peak of a CY5 channel; absence of a high Tm melting peak, indicating SMN1-Exon8 double copy deletion; the absence of a low Tm melting peak indicates that SMN2-Exon8 double copies are absent.

ROX fluorescence channel:

the high Tm melting peak is SMN1-Target, and the low Tm melting peak is SMN1-Ref;

when the normal sample SMN1 is not deleted, signals exist in both a high Tm melting peak and a low Tm melting peak of the ROX channel;

when SMN1 double copies are deleted, the ROX channel high Tm melting peak is deleted;

when SMN1 is deleted in a single copy (carrier), the strength Ratio of a high Tm melting peak to a low Tm melting peak is changed, rm (SMN 1-Target) is the strength of the high Tm melting peak, rm (SMN 1-Ref) is the strength of the low Tm melting peak, ROX Ratio = Rm (SMN 1-Target)/Rm (SMN 1-Ref); when the ROX Ratio is more than or equal to 0.75 and less than or equal to 1.05, the copy number of the SMN1 exon7 region is 1 copy; when the ROX Ratio is more than or equal to 1.45 and more than or equal to 1.2, the copy number of the SMN1 exon7 region is 2; when the ROX Ratio is between 1.05-1.2, the copy number analysis result is located in the Gray zone (Gray zone), and the sample needs to be re-detected.

Example 2 optimization of primers (exemplary results are provided for SMN2 Gene interference resistance)

Because the SMN1 gene and the SMN2 gene are highly homologous, in order to avoid the interference of the SMN2 gene, a retardant primer is designed according to the different sites INS7+100 of the SMN1 gene and the SMN2 gene (the SMN1 is INS7+100A, and the SMN2 is INS7+ 100G), and the 3' end of the primer is 1 base different from the SMN1 gene and 2 bases different from the SMN2 gene. And designing a blocking probe, wherein the blocking probe covers the INS7+100 locus and the 3' end position of the primer, the blocking probe is completely matched with the INS7+100G of the SMN2 gene, and the base of the INS7+100G is modified by locked nucleotide, so that the blocking probe is ensured to be preferentially combined with the SMN2 gene, and the interference of the SMN2 gene is avoided.

A downstream primer is designed aiming at a difference locus INS7+100 and is reversely complementary with an original sequence, the last 3 base of a reverse primer R3 at the 3 'end is changed from wild type T to C, so that the amplification difference of an SMN1 gene and an SMN2 gene is increased, one base difference exists between the reverse primer R3 and the SMN1 gene, and two base differences exist between the reverse primer R3 and the SMN2 gene at the 3' end, namely the last 3 base C and the last base T, so that the amplification efficiency of the SMN2 gene can be greatly reduced, and the interference of the SMN2 gene is prevented.

The control primers are reverse primer R3b and reverse primer R3c. The reverse primer R3b only has difference between the SMN1 gene and the SMN2 gene at the INS7+100 site and the last base T. The reverse primer R3c reduces one base T at the 3' end and can be simultaneously combined with the SMN1 gene and the SMN2 gene for amplification.

R3：5'-cggTGTTTTACATTAACCTTTCAACTcTT-3'。

R3b：5'-cggTGTTTTACATTAACCTTTCAACTTTT-3'。

R3c：5'-cggTGTTTTACATTAACCTTTCAACTTT-3'。

6 experimental groups are set for testing the anti-interference effect of the blocking primer and the blocking probe designed for comparison on the SMN2 gene.

The test groups 1-3 added probe FB1 against SMN2 gene (see Table 3 for probe FB 1), and the test groups 4-6 did not add probe FB1. The recipes for the amplification reaction solution and the pretreatment solution are given in step three of example 1. The preparation method of the gene detection solution is shown in Table 11.

The preparation method of the sample template solution comprises the following steps: adding 100 mul of pretreatment liquid into 3 mul of whole blood of a subject, incubating for 10min at 95 ℃, centrifuging for 5min at 2500rpm, and collecting supernatant, namely sample template solution. The subjects were diagnosed as SMN1 double-copy deletant (patient), SMN1 single-copy deletant (carrier), and SMN1 normal, and represent samples of 0 copy, 1 copy, and 2 copies of exon7 of the SMN1 gene, respectively. Mixing 6.0 mul of amplification reaction solution, 2 mul of sample template solution and gene detection solution, adding deionized water to 25 mul, centrifuging to remove bubbles, and obtaining the sample detection reaction system.

Detection was performed according to 3 of step four of example 1.

TABLE 11 Gene test fluid composition

For the SMN1 gene exon 70 copy sample (patient), because only SMN2 exon7 is present, the SMN2 exon signal was amplified with primer R3c, and the SMN1-Target signal peak of the R3 and R3b amplification reactions represents the SMN2 exon non-specific amplification signal.

The results are shown in Table 12 and FIG. 2. As a result, only the blocking primer was used, and still some amplification of the SMN2 gene was observed. The combination of the R3 primer and the blocking probe used in the experimental group 1 can effectively inhibit the amplification of the SMN2, and the inhibition efficiency reaches 99.65%. The inhibition efficiency of the experiment group 4 on the SMN2 gene amplification by using the blocking primer R3 only is 80.88%. In the experimental group 2, the combination of the primer R3b and the blocking probe is used, and the inhibition efficiency of the SMN2 gene amplification is 70.72%. In experiment group 5, the inhibition efficiency of the primer R3b alone on the SMN2 gene amplification is 50.98%. Therefore, the blocking primer and the blocking probe used in the invention can effectively eliminate the interference of SMN2 gene amplification and improve the specificity and accuracy of detection.

TABLE 12

Example 3 Point mutation detection Probe System screening

Control probes were set and several adjacent quenching probe sets were changed to self-quenching probes. The information regarding the control probes and the nucleotide sequences are shown in Table 13.

TABLE 13 information relating to control probes and nucleotide sequences

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The composition of control Gene assay solution A for 1 reaction (total volume 6. Mu.l) is shown in Table 14. The primers and probes in the gene detection solution A are used for detecting c.22dupA, c.863G > T, c.400G > A and c.463_464delAA. That is, the probes were replaced in the same manner as the primers used in Table 5.

The composition of control Gene assay solution B for 1 reaction (total volume 6. Mu.l) is shown in Table 15. The primers and probes in the gene detection solution B were used for detection of 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 probes were replaced in the same manner as the primers used in Table 6.

TABLE 14

Watch 15

The detection sample is a normal newborn dry blood spot sample.

The kit prepared in step three of example 1 was used and detection was performed in accordance with step four of example 1 (using only gene detection solution A and gene detection solution B).

The detection was performed by replacing the gene assaying solution A with the control gene assaying solution A and replacing the gene assaying solution B with the control gene assaying solution B, according to the fourth step of example 1.

As a result, as shown in FIG. 3, the signal intensity of the neighboring quenched probe was stronger than that of the self-quenched probe.

Example 4 screening of detection Probe System for exon7 and exon8 of SMN1 Gene

Control probes were set and the set of adjacent quenching probes and the self-quenching probe were interchanged. The information regarding the control probes and the nucleotide sequences are shown in Table 16.

TABLE 16

The composition of the gene test solution C of the control group (1 reaction, total volume 6. Mu.l) is shown in Table 17. The primer and the probe of C in the gene detection solution are used for detecting the exon7 and the exon8 of the SMN1 gene, SMN1-Target and SMN1-Ref.

TABLE 17

The composition of the gene assaying liquid C for the test group (1 reaction, total volume 6. Mu.l) is shown in Table 18.

Watch 18

The subjects were confirmed SMN1 double copy deletant (patient), SMN1 single copy deletant (carrier), and SMN1 normal, and represent samples of 0 copy, 1 copy, and 2 copies of exon7 of SMN1 gene, respectively. The preparation method of the sample template solution comprises the following steps: adding 100 mul of pretreatment liquid into 3 mul of whole blood of a subject, incubating for 10min at 95 ℃, centrifuging for 5min at 2500rpm, and collecting supernatant, namely sample template solution.

The procedure of step three in example 1 was repeated except that the gene testing solution C was replaced. Mixing 6.0 mul of amplification reaction solution, 2 mul of sample template solution and gene detection solution C, adding deionized water to 25 mul, centrifuging to remove bubbles, and obtaining the sample detection reaction system. Detection was performed according to 3 of step four of example 1.

The results show that in the SMN1 exon7 shown in FIG. 4 and FIG. 5, the signal of the adjacent quenching dual-probe system is stronger than the fluorescent signal of the self-quenching system probe, and the distinguishing effect on

copy numbers

0,1 and 2 is similar. SMN1 exon8, a double-probe system signal is similar to a self-quenching system probe fluorescent signal, but the distinguishing effect of the adjacent quenching double-probe system on

exon8 copy numbers

1 and 2 is obviously better than that of the self-quenching probe system. For the SMN1-Target region, the probe signal of the self-quenching probe system is stronger than that of the adjacent double-probe system, and the result is shown in FIG. 6.

Example 5 plasmid detection

This example relates to 11 plasmids, in the order plasmids Z1 to Z11. The plasmid-related information is shown in Table 19, and contains the wild-type or mutant gene sequences of the SMN1 gene loci to be tested. The plasmid was prepared by Biotechnology engineering (Shanghai) GmbH: synthesizing DNA molecules corresponding to each gene locus by adopting a gene synthesis mode, and then cloning the DNA molecules onto the PUC-57 plasmid.

TABLE 19 Gene Synthesis of plasmids containing the respective SMN1 Gene site sequences

Plasmids	Genotype(s)	The foreign DNA molecule used for insertion into the plasmid corresponds to the location of the genome (2013, GRCh38/hg 38)
			Z1	c.22dup (p.Ser8LysfSX23A) homozygous mutation	And (2) Chr5:70924941-70925480, 70925125 insertion A
Z2	c.683T>A (p.Leu228X) homozygous mutations	And (2) Chr5:70944501-70944980, 70944713 base T mutated to A
			Z3	c.689C>T (p.Ser230Leu) homozygous mutation	And (2) Chr5:70944501-70944980, 70944719 base C mutated to T
Z4	c.863G>T (p.Gly279Glufs 5) homozygous mutationBecome	And (2) Chr5: bases G from 70951701 to 70952190,70951969 are mutated to T
			Z5	c.400G>A (p.Glu134Lys) homozygous mutation	And (2) Chr5: base G of 70942271-70942760,70942482 is mutated to A
Z6	c.835-5T>G (p.Gly279Glufs 5) homozygous mutation	And (2) Chr5: base T of 70951701-70952190,70951936 is mutated to G
			Z7	c.463_464delAA (p.Asn 155Cysfs 5) homozygous mutation	And (2) Chr5: deletion of basic group AA of 70942271-70942760,70942547-70942548
Z8	c.5C>G (p.Ala2Gly) homozygous mutation	And (2) Chr5:70924941-70925480, 70925108 base C mutated to G
			Z9	c.40G>T (p.Glu13X) homozygous mutation	And (2) Chr5:70924941-70925480, 70925143 base G mutated to T
Z10	c.43C>T (p.Glu 14X) homozygous mutation	And (2) Chr5:70924941-70925480, 70925146 base C mutated to T
			Z11	c.56delT(p.Val19Glyfs*21 Homozygous mutation	And (2) Chr5: base T deletions of 70924941-70925480, 70925159

Taking a plasmid (any one of the plasmids Z1 to Z11), and diluting to 0.1 pg/mu l to obtain a simulated homozygous mutation sample.

11 plasmids, and 11 simulated homozygous mutant samples are obtained correspondingly.

The genomic DNA of a normal person (subjected to sequencing verification and each relevant site is wild type) is used as a wild sample.

The results are shown in FIGS. 7 and 8. For comparison, each figure is an overlay of a mock homozygous mutant sample (homozygous mutant) and a wild sample (wild type). The result shows that the kit provided by the invention can effectively detect the wild type and the homozygous mutant type of the gene locus to be detected.

Example 6 sensitivity test

Genomic DNA of normal persons (subjected to sequencing verification, and all relevant sites are wild type) is extracted. The genomic DNA was subjected to gradient dilution and used as a sample template solution.

The kit prepared in step three of example 1 was used and the detection was performed according to step four of example 1. The amount of the genomic DNA added as a template in each detection reaction system was set to 10ng, 5ng, 2.5ng, 1.25ng, 0.675ng, 0.338ng or 0.168ng, respectively.

The results show that the signal is still good as low as 1.25ng addition. Therefore, the detection sensitivity was set to 2 ng/25. Mu.l reaction system, ensuring the detection signal quality.

Example 7 clinical sample validation

The subjects of this example were 16 patients with spinal muscular atrophy who had been diagnosed.

And collecting anticoagulated blood by a subject, and preparing a dry blood spot sample. The kit prepared in step three of example 1 was used and the detection was performed according to step four of example 1.

Subjects collected anticoagulated blood and blood DNA was purified using a tiangen genomic purification kit for MLPA validation and Sanger sequencing validation.

Among 16 clinical spinal muscular atrophy samples, 13 samples were detected as SMN1 gene exon7 biallelic homozygous deletion, and 3 samples were detected as SMN1 gene complex heterozygous mutation, i.e. one allele deletion and the other allele having minor pathogenic variation (including point mutation).

The detection result of the kit is consistent with the MLPA and sequencing verification results.

Example 8 validation of clinical samples

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

The kit can detect a patient with double allelic homozygous deletion of the SMN1 gene exon7, an exemplary detection effect graph is shown in figure 9, a melting peak representing SMN1-exon7 has no signal, only a melting peak of SMN2-exon7 appears in a FAM fluorescence channel in a detection system C, and a melting peak appears in SMN1-Ref and no melting peak signal exists in SMN1-Target in an ROX fluorescence channel.

The kit can detect carriers with single copy deletion of exon7 of SMN1 gene, and an exemplary detection effect graph is shown in figure 10.

The kit can detect normal samples (double copies of exon7 of SMN1 gene), and an exemplary detection effect graph is shown in FIG. 11.

And (3) the ratio of melting peaks of the ROX fluorescence channel in the detection system C, the carrier SMN1-Target and SMN1-Ref is reduced, and the quantitative calculation is shown in figure 12. Detecting whether the copy number of the SMN1 exon7 is 0,1 or 2 through the Ratio of melting peaks of SMN1-Target and SMN1-Ref, wherein different copy numbers of the SMN1 exon7 (0,1,2) can be well distinguished, when SMN1 is lost in a single copy (carrier), the strength Ratio of a high Tm melting peak and a low Tm melting peak is changed, rm (SMN 1-Target) is the strength of the high Tm melting peak, rm (SMN 1-Ref) is the strength of the low Tm melting peak, and ROX Ratio = Rm (SMN 1-Target)/Rm (SMN 1-Ref); when the ROX Ratio is more than or equal to 0.75 and less than or equal to 1.05, the copy number of the SMN1 exon7 region is 1 copy; when the ROX Ratio is more than or equal to 1.45 and more than or equal to 1.2, the copy number of the SMN1 exon7 region is 2.

The kit can detect patients caused by SMN1 gene compound heterozygous mutation, one allele loss passes through an ROX fluorescence channel in a detection system C, the ratio of melting peaks of a carrier SMN1-Target and SMN1-Ref is reduced and determined, allele point mutation detection passes through detection reaction systems A and B, and three compound heterozygous mutant patients are detected to carry common point mutation: c.22dup (p.Ser8LysFSX23A), c.400G > A (p.Glu134Lys), c.863G > T (p.Gly279Glufs. 5). Exemplary results are shown in fig. 13, 14, 15, respectively. For ease of comparison, each figure is an overlay of an exemplary sample.

The kit can accurately detect SMN1 complete deletion samples and single copy deletion samples (carriers), and investigate the interference of common different copy numbers of SMN2 exon7 on SMN1 exon7 copy number detection. Exemplary results are shown in fig. 16.SMN2 exon7 is common with different copy numbers of 0,1,2 and 3, the copy number detection result of SMN1 exon7 is not influenced, and the kit has good anti-interference capability on SMN 2.

The detection results of the kit are consistent with MLPA and sequencing verification results.

Example 9 detection of dried blood Spot sample

The sample to be tested in this example is 80 samples of dried blood spots of newborn infants.

The kit prepared in step three of example 1 was used and detection was performed according to step four of example 1.

And (3) detection results: no SMN1 exon7 homozygous deletion mutation was found, and 2 exon7 heterozygous deletions (carriers) were found.

Example 10 saliva sample testing

The sample to be tested in this example is 10 human saliva samples.

The kit prepared in step three of example 1 was used and the detection was performed according to step four of example 1.

The SMN1 gene detection kit prepared in example 1 is used for detecting a sample to be detected.

And (3) detection results: the detection signal is good, and the pure deletion mutation and the heterozygous deletion (carrier) of the SMN1 exon7 are not found.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.

Claims

1. A kit comprises a gene detection solution A, a gene detection solution B and a gene detection solution C;

the gene detection solution A contains a primer F4, a primer R4, a primer F6, a primer R6, a primer F7, a primer R7, a probe FP4, a probe Q2, a probe FP8, a probe Q5, a probe FP9, a probe FP10 and a probe Q6;

the gene detection solution B contains a primer F4, a primer R4, a primer F6, a primer R6, a primer F5, a primer R5, a probe FP6, a probe Q4, a probe FP7, a probe FP11, a probe Q7, a probe FP5 and a probe Q3;

the gene detection solution C contains a primer F1, a primer R1, a primer F2, a primer R2, a primer F3, a primer R3, a probe FP1, a probe FP2, a probe Q1, a probe FP3 and a probe FB1;

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

probe FP1 is SEQ ID NO:15, a single-stranded DNA molecule; probe FP2 is SEQ ID NO: 16; probe Q1 is SEQ ID NO: 17; probe FP3 is SEQ ID NO:18, a single-stranded DNA molecule; probe FB1 is SEQ ID NO: 19; probe FP4 is SEQ ID NO:20, a single-stranded DNA molecule; probe Q2 is SEQ ID NO:21, a single-stranded DNA molecule; probe FP5 is SEQ ID NO: 22; probe Q3 is SEQ ID NO: 23; probe FP6 is SEQ ID NO:24, a single-stranded DNA molecule; probe Q4 is SEQ ID NO: 25; probe FP7 is SEQ ID NO: 26; probe FP8 is SEQ ID NO:27, a single-stranded DNA molecule; probe Q5 is SEQ ID NO: 28; probe FP9 is SEQ ID NO: 29; probe Q6 is SEQ ID NO: 30; probe FP10 is SEQ ID NO: 31; probe FP11 is SEQ ID NO:32, a single-stranded DNA molecule; probe Q7 is SEQ ID NO:33, or a single-stranded DNA molecule as set forth in fig. 33.

2. The kit of claim 1, wherein: the kit also comprises a pretreatment solution and an amplification reaction solution;

the pretreatment liquid contains Chelex-100, triton-X100 and NaOH;

3. The application of the gene detection solution A, the gene detection solution B and the gene detection solution C in the preparation of the kit;

the gene detection solution A is the gene detection solution A according to claim 1;

the gene assaying solution B is the gene assaying solution B according to claim 1;

the gene assaying solution C according to claim 1.

4. The application of the gene detection solution A, the gene detection solution B, the gene detection solution C, the pretreatment solution and the amplification reaction solution in the preparation of the kit;

the gene assaying solution C according to claim 1.

The pretreatment liquid is the pretreatment liquid according to claim 2;

the amplification reaction solution is the amplification reaction solution according to claim 2.

5. The kit or use according to any one of claims 1 to 4, wherein:

in the gene detection solution A, the molar ratio of a primer F4, a primer R4, a primer F6, a primer R6, a primer F7, a primer R7, a probe FP4, a probe Q2, a probe FP8, a probe Q5, a probe FP9, a probe FP10 and a probe Q6 is as follows in sequence: 1.5:15:12:1.2:1:10:6:6:6:6:5:5:5;

in the gene detection solution B, the molar ratio of a primer F4, a primer R4, a primer F6, a primer R6, a primer F5, a primer R5, a probe FP6, a probe Q4, a probe FP7, a probe FP11, a probe Q7, a probe FP5 and a probe Q3 is as follows in sequence: 1.5:15:1:10:1.5:15:7.5:7.5:7.5:5:5:6:6;

6. the kit or use according to any one of claims 1 to 4, wherein:

BHQ2 is marked at the tail end of the probe Q1 and 3';

the 3,5 'end of the probe FP is marked with ROX, and the 3' end is marked with BHQ2;

marking CY5 and 3 'ends at the 4,5' ends of the probes FP for C3Spacer modification;

the BHQ2 is marked at the tail end of the probe Q2 and 3';

the end of the probe Q3,3' is marked with BHQ2;

labeling FAM at the 3' end of the probe FP 6;

the probe FP8,5 'end marks ROX, and the 3' end is modified by C3 Spacer;

the MGB is marked at the 5,3' end of the probe Q;

labeling FAM at the 3' end of the probe FP 9;

the 3' end of probe Q7 is labeled with MGB.

7. The primer probe group consists of a primer F1, a primer R1, a primer F2, a primer R2, a primer F3, a primer R3, a primer F4, a primer R4, a primer F5, a primer R5, a primer F6, a primer R6, a primer F7, a primer R7, a probe FP1, a probe FP2, a probe Q1, a probe FP3, a probe FB1, a probe FP4, a probe Q2, a probe FP5, a probe Q3, a probe FP6, a probe Q4, a probe FP7, a probe FP8, a probe Q5, a probe FP9, a probe Q6, a probe FP10, a probe FP11 and a probe Q7;

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

probe FP1 is SEQ ID NO:15, a single-stranded DNA molecule; probe FP2 is SEQ ID NO: 16; probe Q1 is SEQ ID NO: 17; probe FP3 is SEQ ID NO:18, a single-stranded DNA molecule; probe FB1 is SEQ ID NO: 19; probe FP4 is SEQ ID NO:20, a single-stranded DNA molecule; probe Q2 is SEQ ID NO:21, a single-stranded DNA molecule; probe FP5 is SEQ ID NO: 22; probe Q3 is SEQ ID NO: 23; probe FP6 is SEQ ID NO: 24; probe Q4 is SEQ ID NO: 25; probe FP7 is SEQ ID NO: 26; probe FP8 is SEQ ID NO:27, a single-stranded DNA molecule; probe Q5 is SEQ ID NO: 28; probe FP9 is SEQ ID NO: 29; probe Q6 is SEQ ID NO: 30; probe FP10 is SEQ ID NO: 31; probe FP11 is SEQ ID NO:32, a single-stranded DNA molecule; probe Q7 is SEQ ID NO:33, or a single-stranded DNA molecule as set forth in fig. 33.

8. The primer probe set of claim 7, wherein:

the end of the probe Q1,3' is marked with BHQ2;

probe FP3,5 'end mark ROX,3' end mark BHQ2;

the end of the probe Q2,3' is marked with BHQ2;

the end of the probe Q3,3' is marked with BHQ2;

FAM is marked at the 3' end of the probe FP 6;

the MGB is marked at the 5,3' end of the probe Q;

labeling FAM at the 3' end of the probe FP 9;

marking CY5 at the 5 'end of the probe FP11, and modifying the 3' end by using a C3 Spacer;

the 3' end of probe Q7 is marked with MGB.

9. Use of a primer probe set according to claim 7 or 8 for the preparation of a kit.

10. A kit comprising the primer probe set of claim 7 or 8.