CN103614477B - Fluorescent quantitative PCR (Polymerase Chain Reaction) kit for diagnosing human spinal muscular atrophy - Google Patents

Abstract

The invention relates to a fluorescent quantitative PCR kit for diagnosing human spinal muscular atrophy. The kit includes amplification primers and fluorescent probes, and is characterized in that the amplification primers are a pair of specifically amplified SMN1 and a shared primer of the SMN2 gene, a pair of primers for specifically amplifying the NAIP gene, and a pair of primers for specifically amplifying the reference sequence; the fluorescent probe is a fluorescent probe for specifically detecting the SMN1 gene, a specific A fluorescent probe for detecting the SMN2 gene, a fluorescent probe for specifically detecting the NAIP gene, and a fluorescent probe for specifically detecting a reference sequence; the reference sequence is the sequence shown in SEQ NO.14, which is located at the end of the NAIP gene GRCh37/hg19chr5 at 78kb on the grain side: 71107506-71107618 interval. The kit of the invention uses the DNA sequence located at the telomeric side of the NAIP gene as a reference sequence, which significantly improves the accuracy and stability of diagnosis.

Description

A fluorescent quantitative PCR kit for diagnosing human spinal muscular atrophy

technical field

The invention relates to the field of biochemistry, in particular to reagents for nucleic acid determination or detection methods.

Background technique

Spinal muscular atrophy (SMA) is a group of common autosomal recessive genetic diseases, ranking second among fatal autosomal recessive genetic diseases, and the incidence rate of patients in live births is 1/6000~ 1/10000. Clinically, SMA is generally divided into 4 types: Type Ⅰ (also known as Werding-Hoffman disease) is the most severe subtype (severe), accounting for about half of SMA patients, with acute onset and rapid progression, usually within 6 months of birth Onset within 2 years; SMA type Ⅱ is chronic infantile type (intermediate type), usually onset within 7 to 18 months, and most of them can survive 10 to 20 years old; SMA type Ⅲ (also known as Wohlfart-Kugelberg- Welander disease) is juvenile type (mild type), and the onset occurs after 18 months of birth. The disease develops slowly and can generally survive to adulthood. Most of them die due to respiratory muscle paralysis or systemic failure; SMA IV type is adult type (very mild type), The onset usually occurs after the age of 20 to 30, and the main manifestations are slow and gradual proximal weakness and muscle atrophy of the upper and lower extremities, and can walk in adulthood. In general, SMA is a fatal disease, a severe neuromuscular disease, and currently there is no effective treatment in clinical practice.

Under normal circumstances, the human body will normally express SMN protein and NAIP protein to maintain the function of spinal cord anterior horn cells. When the expression of SMN protein and NAIP protein decreases or even disappears, it will lead to the degeneration of the anterior horn cells of the spinal cord, resulting in progressive muscle weakness and muscle atrophy of the individual trunk and proximal limb skeletal muscles, that is, spinal muscular atrophy (SMA). The etiology of SMA is due to the deletion or mutation of SMA-related genes (mainly including SMN gene and NAIP gene), resulting in the decrease or even disappearance of SMN protein and NAIP protein expression. The main method of diagnosis.

Human SMA-related genes are located on the long arm of chromosome 5, zone 1, zone 3, zone 2 (5q13.2), including the spinal cord motor neuron (Spinal Motor Neurons, SMN) gene and neuronal apoptosis inhibitory protein (Neuronal Apoptosis Inhibitory Protein, NAIP) gene. Among them, the SMN gene is 20kb in full length, including 9 exons and 8 introns, and has two very similar gene copies, SMN1 (or SMNt) located at the telomere end and SMN2 (or SMN2) located at the centromere end. SMNc). SMN1 and SMN2 are two highly homologous inverted repeat DNA sequences, with only 5 base differences at the 3' end, especially the single base difference between SMN2 and SMN1 due to the 7th exon (c.840C> T), resulting in different protein products encoded by the two gene copies, that is, SMN1 encodes a complete and stable SMN functional protein, while SMN2 mainly encodes a truncated SMN protein with functional defects, and a small part (10%-50%) encodes a complete For the stable SMN functional protein, when the SMN1 gene function is completely lost, the expression of the SMN2 gene has a certain supplementary effect on the function of the SMN protein, and is defined as a modifier gene of SMA. The full length of the NAIP gene is 70kb, including 16 exons and 15 introns. The encoded NAIP protein inhibits nerve cell apoptosis by blocking the activation of caspase-3 and caspase-7 in the cell signal transduction pathway. NAIP protein Loss of function leads to excessive apoptosis of motor cells in the anterior horn, damages motor neurons, causes secondary muscle atrophy, and aggravates the condition of SMA. Therefore, NAIP gene is also defined as a modifier gene of SMA.

Current research has shown that the etiology of SMA is mainly due to the partial sequence deletion of SMN gene and NAIP gene: among them, SMN1 is the determining gene, which expresses a complete and stable SMN functional protein, and SMN2, as a modifier gene of SMA, expresses a biologically active SMN protein Although the expression amount is small, with the increase of its copy number, there will be a cumulative effect of the expression amount, so that the patient's clinical symptoms can be alleviated to a certain extent. Regarding the NAIP gene, studies have shown that its deletion (the deletion range is exon 5 and 6) is related to the severity of SMA, and the clinical phenotype of SMA patients involving NAIP gene deletion is severe, and only a single SMN gene deletion is not involved in NAIP SMA patients with gene deletion have mild clinical phenotype. According to reports, 98.7% (226/229) of the children with SMN1 gene deletions, about 90% of SMA patients showed homozygous SMN1 exon 7 and/or 8 deletions. Iannaccone et al. reported that due to gene transformation, the gene copy number of SMN2 in the population is 0-4, not just 2 copies. This project investigated the copy number of SMN2 in 1712 Chinese newborns, and found that the imbalance of SMN2 gene copy number in the Chinese population is a common phenomenon. Accordingly, homozygous deletion of SMN1 gene sequence leading to loss of SMN protein function is a diagnostic indicator for SMA, and the functional status of SMN2 and NAIP genes affects the severity of the clinical phenotype of the disease. Therefore, simultaneous detection of the deletion or multiple copies of the target fragments of SMN1 and NAIP can not only accurately diagnose SMA, but also is necessary for analyzing and estimating the clinical phenotype of SMA.

According to the molecular mechanism of SMA, the current diagnostic strategy is to conduct routine and prenatal genetic diagnosis by analyzing whether the target segment of the SMN1 gene is missing and the number of deletions. With the development of molecular biology techniques, different molecular diagnostic techniques for SMA have appeared successively. Including some traditional methods such as PCR-single-strand conformation polymorphism analysis, single-base mutation PCR technology and denaturing high-performance liquid chromatography to analyze SMN gene exon 7 and 8 mutations. These techniques generally have disadvantages such as low accuracy rate, complicated and time-consuming operation, high cost of detection reagents and expensive instruments and equipment. Since then, the application of real-time fluorescent quantitative PCR technology to detect the deletion of SMN1 gene has appeared. At present, these technical methods are all based on the detection of the deletion of the target sequence of the SMN1 gene, and the deletion or multi-copy analysis of the target sequence of the SMN2 gene and NAIP gene is not included in the detection scope, and there is no comprehensive detection and analysis based on the SMN1/2 and NAIP gene. This situation is mainly caused by the following three aspects: one is that the important role of SMN2 gene and NAIP gene in SMA clinical classification is not realized, and the other is that SMN2 gene is highly homologous to SMN1 gene sequence, only 5 base The differences in bases and the detection of SMN1 and SMN2 genes at the same time put forward higher requirements for the technology. Third, when the number of TaqMan probes is increased in the same system, it will increase the difficulty of system optimization and detection. Moreover, if only the SMN1 gene sequence is used for detection, false positive detection results often occur due to the high similarity between the SMN1 gene and SMN2 gene sequences. In addition, due to the inappropriate selection of reference sequences in fluorescent quantitative detection, the detection results are greatly affected by the DNA extraction method, and the repeatability and accuracy of the detection results are poor. It is difficult to form a kit for clinical promotion. Therefore, it is very necessary to select an appropriate reference sequence and use technical means to detect the SMN1 gene sequence, SMN2 gene sequence and NAIP gene sequence, both in terms of clinical practical application and detection technical requirements. At present, the routine detection of SMA in clinical practice mainly adopts the Multiple Ligation-dependent Probe Amplification (MLPA) detection kit from MRC Company in the Netherlands. ), requires specific analytical instruments, takes a long time (about 24 hours), and has a heavy burden on patients, making it difficult to carry out large-scale population screening and technology promotion in clinical practice.

With the successive implementation of prevention plans and measures aimed at reducing the birth rate of SMA children, as well as in-depth research on the molecular mechanism of SMA pathogenesis and molecular epidemiology, the research and development is accurate, reliable, simple and practical, capable of automation and standardization, and suitable for large-scale populations The high-throughput of screening and routine molecular diagnosis can quickly detect the status of the copy number of SMN1, SMN2 and NAIP gene sequence at the same time, so that a new molecular diagnostic kit for the diagnosis of SMA is currently urgently needed.

Contents of the invention

The technical problem to be solved by the present invention is to provide a fluorescent quantitative PCR kit for diagnosing human spinal muscular atrophy, which has the advantages of accurate and stable diagnostic results.

The scheme that the present invention solves the problems of the technologies described above is as follows:

A fluorescent quantitative PCR kit for diagnosing human spinal muscular atrophy, the kit includes amplification primers and fluorescent probes, characterized in that the amplification primers are a pair of specifically amplified SMN1 and SMN2 genes common primers, a pair of primers for specifically amplifying the NAIP gene, and a pair of primers for specifically amplifying the reference sequence; the fluorescent probes are fluorescent probes for specifically detecting the SMN1 gene, and specifically detecting the SMN2 gene Fluorescent probes for specific detection of NAIP genes and fluorescent probes for specific detection of reference sequences; wherein,

(1) Among the pair of shared primers for specifically amplifying SMN1 and SMN2 genes,

The upstream primer sequence is: 5'-CATCCATATAAAGCTATCTATATAG-3' (SEQ NO.1),

The downstream primer sequence is: 5'-CTTAATTTAAGGAATGTGAGCACCT-3' (SEQ NO.2);

(2) Among the pair of primers for specifically amplifying the NAIP gene,

The upstream primer sequence is: 5'-TTGTGACTTATGAGCCGTACAGC-3' (SEQ NO.3),

The downstream primer sequence is: 5'-AGGCTACAGCAGAAGCACTGAAT-3' (SEQ NO.4);

(3) Among the pair of primers for specifically amplifying the reference sequence,

The upstream primer sequence is: 5'-GAACACACATGTCAGAAGTCTAAG-3' (SEQ NO.5),

The downstream primer sequence is: 5'-ACCTCCAGTTAGATCTTCACTTCT-3' (SEQ NO.6);

(4) The fluorescent probe for specifically detecting the SMN1 gene is:

5'-TTTGATTTTGTCTGAAACCCTGTAAGG-3' (SEQ NO.7), the fluorescein and its quenching group of the probe are 5'-HEX, 3'-BHQ1;

(5) The fluorescent probe for specifically detecting the SMN2 gene is:

5'-TTTTGATTTTGTCTAAAACCCTGTAAGGA-3' (SEQ NO.8), the fluorescein and its quenching groups of the probe are 5'-FAM, 3'-BHQ1;

(6) The fluorescent probe for specifically detecting the NAIP gene is:

5'-ATGGATACCACAGGAGATGGCG-3' (SEQ NO.9), the fluorescein and its quenching group of the probe are 5'-CY5, 3'-BHQ2;

(7) The fluorescent probe for specifically detecting the reference sequence is:

5'-CAAATGCATCAGATTCCCACAAGCT-3' (SEQ NO.10), the fluorescein and its quenching group of the probe are 5'-ROX, 3'-BHQ2.

（8）所述的参比序列为acctccagtt agatcttcac ttctaaagct aagtgagacc tgagacccatgtagacaccc aagaagcttg tggaatctga tgcatttggc ttagacttct gacatgtgtg ttc（SEQ NO.14），该序列位于NAIP基因端粒侧78kb处的GRCh37/hg19chr5:71107506－71107618区间。

The amplification primers and fluorescent probes of the present invention can be synthesized by methods commonly used in the art.

In the fluorescent probe of the present invention, HEX refers to hexachloro-6-methylfluorescein, FAM refers to carboxyfluorescein, CY5 refers to cyanine dye molecule 5, ROX refers to carboxy-X-rhodamine, and BHQ1 and BHQ2 are Refers to the fluorescence quencher group.

The fluorescent quantitative PCR kit of the present invention also includes common reagents for multiplex PCR, such as DNA polymerase, 50-200ng/ul normal genotype control gDNA specimen, magnesium dichloride and dNTPs, wherein the DNA polymerase is Ex Taq. DNA polymerase, magnesium dichloride and dNTPs were purchased from TaKaRa Dalian Co., Ltd.

In the fluorescent quantitative PCR kit of the present invention, the target genes SMN1, SMN2 and NAIP and the reference sequence are located on the long arm of chromosome 5, zone 1, zone 3, zone 2 (5q13.2), and the length of the amplicon DNA sequence is They are all 80-150bp, GC content 20%-60%, no consecutive 5 G or C (see Figure 3).

The position information of the DNA sequence (SEQ NO.1-10) in the fluorescent quantitative PCR kit of the present invention is shown in the following table:

The using method of test kit of the present invention is:

(1) The reaction system is shown in Table 1 below:

Table 1 PCR reaction system of the kit of the present invention

The specific operation method during detection is as follows: each time, according to the amount of specimens to be tested, take a corresponding number of eight-connected PCR reaction tubes, calculate the total amount required for each component according to the total amount of PCR reaction, and then mix the prepared reaction tubes. The solution was divided into a single PCR reaction tube with eight tubes, and finally the template gDNA was added, and the corresponding eight-connected PCR reaction tube was capped, centrifuged at 8000rpm for 30 seconds, and placed in a fluorescent quantitative PCR amplification instrument. The reaction tubes used in the reaction were imported eight-connected PCR tubes (GE-BIO ^TM ), which were purchased from Guangzhou Haoyang Trading Co., Ltd. Wherein, the preparation method of the reaction mixture is as follows: first mix the SMN1 and SMN2 gene shared primers, NAIP gene primers and reference sequence primers according to the ratio of 2/1/1; then mix the SMN1 gene probe, SMN2 gene probe The needle, NAIP gene probe and reference sequence probe were mixed according to the ratio of 1/1/1/1, and then added to the reaction system except the template gDNA and other components.

(2) The reaction program is: pre-denaturation at 95°C for 5 min; 30 cycles at 95°C for 30 sec+60°C for 1 min, and 35 cycles. At the end of the annealing step at 60°C, the fluorescent signals of HEX, FAM, CY5, and ROX were collected, representing SMN1, SMN2, NAIP, and reference sequence.

The specific operation process is as follows: On the fluorescent quantitative PCR instrument, select the position of the desired detection reaction tube, check the four fluorescent markers respectively HEX, FAM, CY5, and ROX, and number the reaction tubes in sequence. Amplification detection conditions are the reaction program setting, saving the file, and running.

The instrument used for the reaction was Stratagene Mx3005P, which was purchased from Stratagene Corporation of the United States.

(3) Sample processing: gDNA samples can be obtained by the following methods: extract peripheral whole blood samples, anticoagulate with EDTA, and extract gDNA samples by conventional phenol-chloroform extraction.

(4) Sample detection: use gDNA samples to be tested, 2-3 normal gDNA samples with 2 copies of SMN1, SMN2 and NAIP genes as reference samples, and 1-2 copies of SMN1, SMN2 and NAIP genes with known copy numbers Using the above reaction system and reaction procedures, carry out amplification detection on a fluorescent quantitative PCR instrument, and record the Ct value (C stands for Cycle, t stands for threshold, and the meaning of Ct value is the arrival of the fluorescent signal in each reaction tube. The number of cycles experienced when the threshold value is set, and the size of its value reflects the number of templates detected).

Setting of analysis conditions: The system automatically adjusts the threshold value of each fluorescence channel according to the image after analysis (the general area is in the index area). Then export the Ct values of the four fluorescent signals of HEX, FAM, CY5, and ROX (representing SMN1, SMN2, NAIP, and the reference sequence respectively) in each detection tube to an Excel file.

(5) Data analysis and result judgment: refer to the basic model of the literature (Kenneth J, et al.METHODS.2001;25:402–408.), using the relative quantitative method of the ΔCt value method, which is based on the Ct value of each template The logarithmic linear relationship with the initial copy number of the template, the higher the initial copy number, the smaller the Ct value. The principle of the ΔCt value method is to compare the difference between the Ct value of the target gene and the reference sequence (ie ΔCt value) in normal people. If one of the target genes is missing, compared with normal diploids, the amount of the target gene will decrease and the Ct value will increase. , the ΔCt value between it and the reference sequence will increase. Therefore, the original copy number of the target gene of the unknown sample can be judged by comparing the ΔCt value of gDNA of different samples to be tested and the change of ΔCt value of gDNA of normal samples. .

According to the Ct value obtained by quantitative detection, the normal samples with 2 copies of SMN1, SMN2 and NAIP genes were used as control samples. First, for all samples to be tested and control samples, use the Ct value of the reference sequence to normalize the Ct value of the target gene, and calculate according to the following formula:

The target gene of the sample to be tested ΔCt_sample _{to be} tested=Ct_target _gene -Ct_reference _sequence

Control sample target gene ΔCt_control _sample =Ct_target _gene -Ct_reference _sequence

Secondly, compare the ΔCt value of the sample to be tested with the ΔCt value of the control sample (i.e. ΔΔCt value):

The target gene of the sample to be tested ΔΔCt = ΔCt_sample _{to be} tested-ΔCt_control _sample

Finally, calculate the ratio of expression levels: 2 - ^ΔΔCt = ratio of expression

The relative copy number of the target gene of the sample to be tested is Cs=2 ^-ΔΔCt ×2 (2 is the control sample, and the copy number of the target gene is known).

The result obtained is the multiple of the increase or decrease of the target gene in the sample to be tested relative to the control sample calibrated by the expression level of the reference sequence. The purpose of calibrating the expression of the target gene with the reference sequence is to compensate for the difference in amplification efficiency between samples.

In normal individuals, there are 2 copies of SMN1, SMN2, NAIP and the reference sequence. The copy numbers of the three target genes and the reference sequence are equal in quantitative detection, and the ΔCt value is constant (ΔΔCt=0), and the 2 ^-ΔΔCt value is 1.0 ; If one of the three target genes of an individual is missing, the Ct value of this gene increases by 1 during quantitative detection, and the ΔCt value obtained by comparing with the reference sequence also increases by 1 (ΔΔCt=1), the calculated 2 ^-ΔΔCt value is 0.5, which means that the copy number of the target gene is only half of the reference sequence; on the contrary, if a certain target gene has 3 copies, the difference in Ct value of this gene during quantitative detection is 0.58, and the resulting ΔCt change is calculated The 2- ^ΔΔCt value for is 1.42; if both are missing, there will be no amplification of the target sequence. Therefore, according to this value change rule, the relative quantitative method of ΔCt value method was used to directly analyze the relative copy numbers of the target sequences of SMN1, SMN2 and NAIP genes, so as to realize the rapid molecular diagnosis of deletion SMA (see Figure 5).

Normal gDNA samples with 2 copies of SMN1, SMN2 and NAIP genes were used as normal control samples. See Table 2 and Table 3 for the principle of result determination and common genotypes.

Table 2 Judgment of relative copy number quantitative data analysis results

Remarks: If the 2 ^-ΔΔCt value is 0.10~0.3. or 0.70~0.80 or 1.20~1.30, this is the critical value and must be tested again.

Table 3 Common SMA genotypes SMN1, SMN2, NAIP gene relative copy number

The basic principle of the present invention is based on the SMN1 gene, the SMN2 gene and the NAIP gene are all located at 5q13.2, and selecting the adjacent sequence SEQ NO.14 of the same chromosome as the reference sequence can well avoid the reference sequence and purpose caused by DNA extraction The difference in the copy number of the sequence, so as to accurately quantify the copy number of the target gene. At the same time, as far as a normal human genome is concerned (that is, the nuclear genome of a cell), there are two copies of SMN1, SMN2, and NAIP genes each. Deletion-type SMA is caused by the deletion of the SMN1 gene, while SMN2 and NAIP genes have deletions Or multiple copies affect its phenotype. Based on this molecular mechanism, the ultimate goal of any molecular diagnostic method is to determine the exact value of the SMN1 gene copy number, thereby accurately diagnosing SMA. At the same time, by accurately analyzing the multiple copies of the SMN2 gene and the deletion of the NAIP gene, the clinical phenotype of SMA can be estimated. The kit of the present invention adopts the quadruple TaqMan fluorescent quantitative PCR technology to quantitatively detect the copy numbers of SMN1, SMN2 and NAIP genes in the sample, and realizes the rapid molecular diagnosis of SMA by the change of the copy numbers.

During the DNA extraction process, due to the difference in the size of the broken fragments of different chromosomes, the original copy number of the reference sequence located on different chromosomes is different, resulting in inaccurate detection results (see Figure 1). The present invention uses the conserved sequence (see Figure 3) SEQ NO.14 located on the telomere side of the NAIP gene and without copy number variation as the reference sequence. Differences in raw copy number due to DNA extraction method (see Figure 2).

Description of drawings

Figure 1 is a schematic diagram of the copy number difference ratio of the original sequence after elution of the reference sequence and the target sequence located on different chromosomes.

Figure 2 is a schematic diagram of the copy number ratio of the original sequence after elution of the reference sequence and the target sequence located on the same chromosome.

Fig. 3 is a detailed position diagram in the genome of each gene primer and probe sequence of the kit of the present invention.

Fig. 4 is a schematic diagram of the positions and related information of representative DNA fragments amplified by the kit of the present invention.

Figure 5 is a graphical representation of data analysis (calculation of 2 ^-ΔΔCt values).

Detailed ways

The common reference gene method described in the following embodiment 1 is the detection method of the fluorescent quantitative PCR kit that adopts commonly used internal reference gene establishment; The adjacent reference sequence method described in the following embodiment 2 is to adopt Detection method of fluorescent quantitative PCR kit.

Example 1 (Analysis of Fluorescence Quantitative Detection Result by Ordinary Reference Gene Method)

1. Reagent composition:

(1) Among the pair of shared primers for specifically amplifying a certain sequence of SMN1 and SMN2 genes,

The upstream primer sequence is: 5'-CATCCATATAAAGCTATCTATATAG-3' (SEQ NO.1),

The downstream primer sequence is: 5'-CTTAATTTAAGGAATGTGAGCACCT-3' (SEQ NO.2);

(2) Among the pair of primers for specifically amplifying the NAIP gene,

The upstream primer sequence is: 5'-TTGTGACTTATGAGCCGTACAGC-3' (SEQ NO.3),

The downstream primer sequence is: 5'-AGGCTACAGCAGAAGCACTGAAT-3' (SEQ NO.4);

(3) In the pair of primers for specifically amplifying a certain sequence of the reference sequence GAPDH gene,

The upstream primer sequence is: 5'-CAGGAGTGAGTGGAAGACAGAAT-3' (SEQ NO.11),

The downstream primer sequence is: 5'-GACCATATTGAGGGACACAAGGT-3' (SEQ NO.12);

(4) The fluorescent probe for specifically detecting a certain sequence of the SMN1 gene is:

5'-TTTGATTTTGTCTGAAACCCTGTAAGG-3'(5'-HEX,3'-BHQ1) (SEQ NO.7);

(5) The fluorescent probe for specifically detecting a certain sequence of the SMN2 gene is:

5'-TTTTGATTTTGTCTAAAACCCTGTAAGGA-3'(5'-FAM,3'-BHQ1) (SEQ NO.8);

(6) The fluorescent probe for specifically detecting the NAIP gene is:

5'-ATGGATACCACAGGAGATGGCG-3' (5'-CY5,3'-BHQ2) (SEQ NO.9);

(7) The fluorescent probe for specific detection of a certain sequence of the reference sequence GAPDH gene is:

5'-CCACACCATCCTAGTTGCCTCCC-3' (5'-ROX, 3'-BHQ2) (SEQ NO. 13).

(8) Other components:

TaKaRa Ex Taq enzyme and matching Buffer, dNTPs, magnesium dichloride (purchased from Takara Company), 2 normal genotype control gDNA specimens with 2 copies of SMN1, SMN2, and NAIP genes.

(9) Genomic DNA extraction reagents: conventional phenol/chloroform method genomic DNA extraction reagents, whole blood column DNA extraction kits from Tiangen Biochemical Technology (Beijing) Co., Ltd.

2. Detection of SMN1 and SMN2 gene copy number:

(1) Source and type of specimen: A total of 100 cases of SMA peripheral blood samples and normal peripheral blood samples with confirmed genotypes were selected from the laboratory specimen bank and collected by cooperative medical institutions, and the genotypes were SMN1/SMN2/NAIP= 15 copies of 2/2/2, 15 copies of SMN1/SMN2/NAIP=2/1/2, 10 copies of SMN1/SMN2/NAIP=2/1/3, SMN1/SMN2/NAIP=2/2/1 A total of 10 copies, SMN1/SMN2/NAIP=2/3/2 a total of 10 copies, SMN1/SMN2/NAIP=1/2/2 a total of 10 copies, SMN1/SMN2/NAIP=1/2/1 a total of 10 copies, SMN1 /SMN2/NAIP=3/1/2, a total of 10 copies, SMN1/SMN2/NAIP=0/2/2, a total of 10 copies.

(2) Genomic DNA was extracted from 100 samples by phenol-chloroform method and column method respectively, and the gDNA samples were diluted to 50-200ng/μl with sterilized double distilled water for later use.

(3) Sample detection: the above-mentioned gDNA samples to be tested, 2 normal control gDNA samples whose gene copy numbers of SMN1, SMN2, and NAIP were all 2, were applied to the above-mentioned reaction system of the present invention (Table 1), and the PCR reaction of the present invention Program (pre-denaturation at 95°C for 5min; 95°C for 30sec+60°C for 1min, 35 cycles, collecting HEX, FAM, and ROX fluorescence signals at the end of the annealing step at 60°C, respectively representing SMN1, SMN2 and the reference sequence), on Stratagene Mx3005P Amplification detection was performed on a fluorescent quantitative PCR instrument, and the Ct value was recorded.

(4) Data analysis and result judgment: Calculate according to the data analysis formula described in the present invention, and determine the target gene copy number obtained by the detection according to the result judgment standard.

3. Test results:

Comparing the target gene copy value deduced from this case with the genotype analysis results of confirmed specimens, both the phenol/chloroform method and the column method have problems such as low accuracy and variability. The detailed results are shown in Table 4. 5.

Table 4 Analysis of detection accuracy and variability of phenol-chloroform-extracted DNA by common reference gene method

Note: V_R: Quantitative detection 2-△△Ct±SD; A: Accuracy; CV: Variation

Table 5 Analysis of detection accuracy and variability of DNA extracted by column method using common reference gene method

Note: V_R: quantitative detection 2- ^△△Ct ± SD; A: accuracy rate; CV: variability

4. Analysis

In the common reference gene method, due to the differences in breakage points of different chromosomes, the size of the genomic DNA fragments formed is not uniform. There are differences in the original copy number, which cannot be effectively referenced, resulting in inaccurate test results. It can be seen from Table 4 and Table 5 that, except for 10 cases of SMN1/SMN2/NAIP=0/2/2SMA patients who could be accurately detected, the accuracy of genotypes in other groups ranged from 30% to 70%. The copy number variation of the target gene deduced from the fluorescence quantitative results was also high, ranging from 6.9% to 76.6%. From the accuracy and stability of this method, it can be seen that in the application of fluorescence quantitative method for detection, the application of common reference gene method cannot accurately diagnose the target gene.

Example 2 (Analysis of Fluorescence Quantitative Detection Result by Proximity to Reference Sequence Method)

1. Reagent composition:

(1) Among the pair of shared primers for specifically amplifying SMN1 and SMN2 genes,

The upstream primer sequence is: 5'-CATCCATATAAAGCTATCTATATAG-3' (SEQ NO.1),

The downstream primer sequence is: 5'-CTTAATTTAAGGAATGTGAGCACCT-3' (SEQ NO.2);

(2) Among the pair of primers for specifically amplifying the NAIP gene,

The upstream primer sequence is: 5'-TTGTGACTTATGAGCCGTACAGC-3' (SEQ NO.3),

The downstream primer sequence is: 5'-AGGCTACAGCAGAAGCACTGAAT-3' (SEQ NO.4);

(3) Among the pair of primers for specifically amplifying the reference sequence,

The upstream primer sequence is: 5'-GAACACACATGTCAGAAGTCTAAG-3' (SEQ NO.5),

The downstream primer sequence is: 5'-ACCTCCAGTTAGATCTTCACTTCT-3' (SEQ NO.6);

(4) The fluorescent probe for specifically detecting the SMN1 gene is:

5'-TTTGATTTTGTCTGAAACCCTGTAAGG-3'(5'-HEX,3'-BHQ1) (SEQ NO.7);

(5) The fluorescent probe for specifically detecting the SMN2 gene is:

5'-TTTTGATTTTGTCTAAAACCCTGTAAGGA-3'(5'-FAM,3'-BHQ1) (SEQ NO.8);

(6) The fluorescent probe for specifically detecting the NAIP gene is:

5'-ATGGATACCACAGGAGATGGCG-3' (5'-CY5,3'-BHQ2) (SEQ NO.9);

(7) The fluorescent probe for specifically detecting the reference sequence is:

5'-CAAATGCATCAGATTCCCACAAGCT-3' (5'-ROX, 3'-BHQ2) (SEQ NO. 10).

(8) Other components:

TaKaRa Ex Taq enzyme and matching Buffer, dNTPs, magnesium dichloride (purchased from Takara Company), 2 normal genotype control gDNA specimens with 2 copies of SMN1, SMN2, and NAIP genes.

(9) Genomic DNA extraction reagents: conventional phenol/chloroform method genomic DNA extraction reagents, whole blood column DNA extraction kits from Tiangen Biochemical Technology (Beijing) Co., Ltd.

2. Detection of SMN1 and SMN2 gene copy number:

(1) Source and type of specimen: A total of 100 cases of SMA peripheral blood samples and normal peripheral blood samples with confirmed genotypes were selected from the laboratory specimen bank and collected by cooperative medical institutions, and the genotypes were SMN1/SMN2/NAIP= 15 copies of 2/2/2, 15 copies of SMN1/SMN2/NAIP=2/1/2, 10 copies of SMN1/SMN2/NAIP=2/1/3, SMN1/SMN2/NAIP=2/2/1 A total of 10 copies, SMN1/SMN2/NAIP=2/3/2 a total of 10 copies, SMN1/SMN2/NAIP=1/2/2 a total of 10 copies, SMN1/SMN2/NAIP=1/2/1 a total of 10 copies, SMN1 /SMN2/NAIP=3/1/2, a total of 10 copies, SMN1/SMN2/NAIP=0/2/2, a total of 10 copies.

(2) Genomic DNA was extracted from 100 samples by phenol-chloroform method and column method respectively, and the gDNA samples were diluted to 50-200ng/μl with sterilized double distilled water for later use.

(3) Sample detection: the above-mentioned gDNA samples to be tested, 2 copies of normal control gDNA samples with gene copy numbers of SMN1, SMN2, and NAIP of 2, were applied to the reaction system of the present invention (Table 1), and the PCR reaction of the present invention Program (pre-denaturation at 95°C for 5min; 95°C for 30sec+60°C for 1min, 35 cycles, collecting HEX, FAM, and ROX fluorescence signals at the end of the annealing step at 60°C, respectively representing SMN1, SMN2 and the reference sequence), on Stratagene Mx3005P Amplification detection was performed on a fluorescent quantitative PCR instrument, and the Ct value was recorded.

(4) Data analysis and result judgment: Calculate according to the data analysis formula described in the present invention, and determine the target gene copy number obtained by the detection according to the result judgment standard.

3. Test results:

Comparing the target gene copy value deduced from this case with the genotype analysis results of confirmed samples, the phenol/chloroform method and the column method have higher accuracy and less variability. The detailed results are shown in Table 6. 7.

Table 6 Analysis of detection accuracy and variability of DNA extracted by phenol-chloroform method using the adjacent reference sequence method

Remarks: V_R: Quantitative detection 2- ^△△Ct ± SD; A: Accuracy; CV: Variation

Table 7 Analysis of the detection accuracy and variability of DNA extracted by the column method using the adjacent reference sequence method

Remarks: V_R: Quantitative detection 2- ^△△Ct ± SD; A: Accuracy; CV: Variation

4. Analysis

In the adjacent reference sequence method, the reference sequence and the target gene are located near the same chromosome. Although there are differences in the sedimentation rates of large and small fragments during the elution process, since the reference sequence is close to the target gene, whether it is Large fragments or small fragments, the difference in original copy number is small, which can effectively avoid the difference in original copy number caused by DNA extraction and elution, and the detection results obtained in this way are very accurate. It can be seen from Table 6 and Table 7 that except for 1 case of SMN1/SMN2/NAIP=2/2/2 and SMN1/SMN2/NAIP=2/1/3 that could not be detected accurately, other types of The accuracy rate of genotype combination detection reached 100%. The copy number variation of the target gene deduced from the fluorescence quantitative results was also small, ranging from 3.3% to 66.7%. From the accuracy and stability of the method, it can be seen that in the fluorescence quantitative method detection, the application of the adjacent reference sequence method can more accurately diagnose the target gene than the common reference gene method.

Example 3 (Sensitivity and Accuracy Small Sample Evaluation of the Kit)

1. The composition of the kit:

(1) Among the pair of shared primers for specifically amplifying a certain sequence of SMN1 and SMN2 genes,

The upstream primer sequence is: 5'-CATCCATATAAAGCTATCTATATAG-3' (SEQ NO.1),

The downstream primer sequence is: 5'-CTTAATTTAAGGAATGTGAGCACCT-3' (SEQ NO.2);

(2) Among the pair of primers for specifically amplifying a certain sequence of the NAIP gene,

The upstream primer sequence is: 5'-TTGTGACTTATGAGCCGTACAGC-3' (SEQ NO.3),

The downstream primer sequence is: 5'-AGGCTACAGCAGAAGCACTGAAT-3' (SEQ NO.4);

(3) In the pair of primers for specifically amplifying a certain segment of the reference sequence,

The upstream primer sequence is: 5'-GAACACACATGTCAGAAGTCTAAG-3' (SEQ NO.5),

The downstream primer sequence is: 5'-ACCTCCAGTTAGATCTTCACTTCT-3' (SEQ NO.6);

(4) The fluorescent probe for specifically detecting a certain sequence of the SMN1 gene is:

5'-TTTGATTTTGTCTGAAACCCTGTAAGG-3'(5'-HEX,3'-BHQ1) (SEQ NO.7);

(5) The fluorescent probe for specifically detecting a certain sequence of the SMN2 gene is:

5'-TTTTGATTTTGTCTAAAACCCTGTAAGGA-3'(5'-FAM,3'-BHQ1) (SEQ NO.8);

(6) The fluorescent probe for specifically detecting a certain sequence of the NAIP gene is:

5'-ATGGATACCACAGGAGATGGCG-3' (5'-CY5,3'-BHQ2) (SEQ NO.9);

(7) The fluorescent probe for specifically detecting a certain sequence of the reference sequence is:

5'-CAAATGCATCAGATTCCCACAAGCT-3' (5'-ROX, 3'-BHQ2) (SEQ NO. 10).

(8) Other components:

TaKaRa Ex Taq enzyme and matching Buffer, dNTPs, magnesium dichloride (purchased from Takara Company), 2 normal genotype control gDNA specimens with 2 copies of SMN1, SMN2, and NAIP genes.

2. Detection of SMN1, SMN2 and NAIP gene copy number:

(1) Specimen source and type: SMA gDNA specimens with confirmed genotypes selected from the laboratory specimen bank and collected by cooperative medical institutions, the genotypes are SMN1/SMN2/NAIP=2/2/2, SMN1/SMN2 /NAIP=0/2/1, SMN1/SMN2/NAIP=0/2/2, SMN1/SMN2/NAIP=0/3/2, SMN1/SMN2/NAIP=1/1/2, SMN1/SMN2/NAIP =1/2/2, SMN1/SMN2/NAIP=1/0/1, SMN1/SMN2/NAIP=2/0/3, SMN1/SMN2/NAIP=2/1/2, SMN1/SMN2/NAIP=3 /2/3 each in 2 copies (20 copies in total), dilute gDNA samples to 50-200ng/μl with sterilized double distilled water for later use.

(2) Sample detection: The above gDNA samples to be tested, 2 normal control gDNA samples with 2 copies of SMN1, SMN2 and NAIP genes, and 2 quality control gDNA samples with known copy numbers of SMN1, SMN2 and NAIP genes , using the above-mentioned reaction system of the present invention (Table 1), with the PCR reaction program of the present invention (pre-denaturation at 95°C for 5 min; 30 cycles at 95°C for 30 sec+60°C for 1 min, 35 cycles, and collecting HEX, FAM, The fluorescent signals of CY5 and ROX, respectively representing SMN1, SMN2, NAIP and the reference sequence), were amplified and detected on the Stratagene Mx3005P fluorescent quantitative PCR instrument, and the Ct value was recorded.

(3) Data analysis and result judgment: Calculate according to the data analysis formula described in the present invention, and determine the target gene copy number obtained by the detection according to the result judgment standard.

3. Test results:

The test results of this case are shown in Table 8. Comparing the target gene copy value deduced according to the SMA genotype of the detected specimen with the results of this case analysis, the sensitivity and accuracy of this kit reached 100%.

Table 8 Relative copy number quantitative data analysis table

Note: Specimen numbers 1 and 2 have no target gene deletion; 3 and 4 are deletions of 2 SMN1 genes and 1 NAIP gene; 5 and 6 are deletions of 2 SMN1 genes; 7 and 8 are deletions of 2 SMN1 genes, and SMN2 genes have 3 copies; 9 and 10 were deletions of 1 SMN1 gene and 1 SMN2 gene; 11 and 12 were deletions of 1 SMN1 gene; 13 and 14 were deletions of 1 SMN1 gene, 2 SMN2 genes and 1 NAIP gene; 15 and 16 was the deletion of 2 SMN2 genes and 3 copies of the NAIP gene; 17 and 18 were the deletion of 1 SMN2 gene; 19 and 20 were the deletions of the SMN1 gene and 3 copies of the NAIP gene.

4. Results analysis and clinical significance:

(1) The results of data analysis showed that both SMN1/2 and NAIP genes had 2 copies, suggesting that the tested specimens did not have the deletion of these three genes, and patients with deletional SMA could be excluded.

(2) The data analysis results showed that there were 2 copies of the SMN2 gene, 1 copy of the NAIP gene, and no SMN1 target gene, suggesting that the tested specimen was an SMA patient with deletion of 2 SMN1 genes and 1 NAIP gene.

(3) The results of data analysis showed that both SMN2 and NAIP genes had 2 copies, and there was no target gene of SMN1, suggesting that the tested specimen was an SMA patient with deletion of 2 SMN1 genes.

(4) The data analysis results showed that there were 3 copies of the SMN2 gene, 2 copies of the NAIP gene, and no SMN1 target gene, suggesting that the tested specimen was an SMA patient with 2 copies of the SMN1 gene missing but 3 copies of the SMN2 gene.

(5) The results of data analysis showed that both SMN1 and SMN2 genes had 1 copy, and NAIP gene had 2 copies, suggesting that the tested specimen was a SMA carrier missing 1 SMN1 gene and 1 SMN2 gene.

(6) The results of data analysis showed that there was one copy of SMN1, and two copies of SMN2 and NAIP genes, suggesting that the tested specimen was a SMA carrier missing one SMN1 gene.

(7) The results of data analysis showed that there was 1 copy of SMN1 gene, 1 copy of NAIP gene, and no target gene of SMN2, suggesting that the tested sample was a SMA carrier missing 1 SMN1 gene, 2 SMN2 genes and 1 NAIP gene .

(8) The results of data analysis showed that there were 2 copies of the SMN1 gene, 3 copies of the NAIP gene, and no SMN2 target gene, suggesting that the tested specimen was missing 2 SMN2 genes, but there were 3 copies of the NAIP gene, and no SMN1 main functional gene Deletion can exclude patients with deletion SMA.

(9) The results of data analysis showed that there were 2 copies of SMN1 and NAIP genes, and 1 copy of SMN2, suggesting that the tested specimen was missing 1 SMN2 gene, and there was no deletion of the main functional gene of SMN1, so patients with deletion SMA could be excluded.

(10) The results of data analysis showed that there were 3 copies of SMN1 and NAIP genes, and 2 copies of SMN2, suggesting that the tested specimens did not have the deletion of these three genes, and patients with deletion SMA could be excluded.

(11) In addition, during data analysis, if a target gene is amplified, but the 2- ^ΔΔCt value is <0.10, it is likely to be caused by non-specific amplification or contamination of trace target gene fragments, which can be judged as No gene for this purpose.

In summary, when the sample to be tested has no SMN1 target gene, it is an SMA patient; when the sample to be tested has 1 SMN1 target gene, it is a SMA carrier; when the sample to be tested has 2 or more SMN1 genes When , patients with deletion-type SMA can be excluded.

Example 4 (Small-scale population screening test evaluation)

1. Specimen collection:

125 peripheral blood samples were randomly collected from the Laboratory Department of the Third Affiliated Hospital of Southern Medical University, and basic information was obtained at the same time. Then use the protease digestion column extraction method developed by our laboratory to extract gDNA, and dissolve the gDNA specimen in sterilized double distilled water for later use.

2. Specimen detection:

(1) Kit composition:

With embodiment 3.

(2) Sample detection: The above 125 gDNA samples to be tested were double-blind, and the MLPA kit detection method and this kit were used for parallel detection. The detection method of this kit is: in each round of PCR reaction, the gDNA specimens to be tested, 2 normal control gDNA specimens with 2 copies of SMN1, SMN2 and NAIP genes, 2 copies of SMN1, SMN2 and NAIP gene copies For known quality control gDNA samples, apply the above-mentioned reaction system of the present invention (Table 1), and use the PCR reaction program of the present invention (pre-denaturation at 95°C for 5 minutes; 95°C for 30sec+60°C for 1min, 35 cycles, annealing step at 60°C Fluorescent signals of HEX, FAM, CY5, and ROX were collected at the end, respectively representing SMN1, SMN2, NAIP, and the reference sequence), amplified and detected on a Stratagene Mx3005P fluorescent quantitative PCR instrument, and the Ct value was recorded.

(3) Data analysis and result judgment: Calculate according to the data analysis formula described in the present invention, and determine the target gene copy number obtained by the detection according to the result judgment standard.

3. Test results:

Both detection methods detected 2 samples with only 1 copy of SMN1 gene deletion, 2 copies of SMN2 and NAIP genes; 1 sample with only 1 copy of SMN1 gene deletion, 2 copies of SMN2 gene, NAIP gene The gene was 1 copy; 3 samples had 3 copies of SMN1 and NAIP genes, and 2 copies of SMN2 gene; 1 sample had 3 copies of SMN1 gene, and 2 copies of SMN2 and NAIP genes; 7 cases The specimens had no target gene of SMN2, and both SMN1 and NAIP genes had 2 copies; 3 samples had no target gene of SMN2, 2 copies of SMN1, and 3 copies of NAIP gene; 22 samples had 1 copy of SMN2 gene, and 2 copies of SMN1 and NAIP All genes have 2 copies; 4 specimens have 3 copies of SMN2 gene, 2 copies of SMN1 and NAIP genes; 1 specimen has 3 copies of SMN2 gene, 2 copies of SMN1 gene, and 1 copy of NAIP gene ; 2 samples had 3 copies of NAIP gene, 2 copies of SMN1 and SMN2. 79 cases had no SMN1/2, NAIP gene deletion and multiple copies, and the coincidence rate of negative and positive was 100%. In these specimens, the copy numbers of SMN1/2 and NAIP genes deduced by the MLPA detection method are consistent with the gene copy number results obtained by the kit of the present invention, with a coincidence rate of 100% (see Table 9).

4. Results analysis and clinical significance:

With embodiment 3.

Table 9 Detection results of SMN1/2 and NAIP gene copy number in 125 random cases

Embodiment 5 (large sample blind analysis and evaluation)

1. Specimen collection:

From the gDNA specimens stored in the sample resource bank of the teaching and research section and cooperative medical institutions, 500 specimens that have been tested by the MLPA method were selected. For this batch of samples, first test their concentration, dilute according to the required sample volume of 30-50 μl and DNA concentration of about 100ng/μl, and then measure the sample concentration on a nucleic acid protein analyzer to ensure that the DNA concentration range is 50-200ng /μl, 1.65﹤OD _260/280 ﹤2.00, the specimens that do not meet the requirements are not included in this evaluation and analysis.

2. Specimen detection:

(1) Kit composition:

With embodiment 3.

(2) Sample detection: After blindly numbering the above 500 gDNA samples to be tested, the kit of the present invention is used for detection. The normal control gDNA samples with SMN2 and NAIP gene copy numbers of 2, 2 copies of SMN1, SMN2 and NAIP gene copy numbers of known quality control gDNA samples, using the above reaction system of the present invention (Table 1), with the PCR of the present invention Reaction program (pre-denaturation at 95°C for 5 minutes; 95°C for 30sec+60°C for 1min, 35 cycles, at the end of the annealing step at 60°C, collect fluorescence signals of HEX, FAM, CY5, and ROX, respectively representing SMN1, SMN2, NAIP, and the reference sequence ), the amplification detection was carried out on the Stratagene Mx3005P fluorescent quantitative PCR instrument, and the Ct value was recorded.

(3) Data analysis and result judgment: Calculate according to the data analysis formula described in the present invention, and determine the target gene copy number obtained by the detection according to the result judgment standard.

(4) Summary analysis and processing of detection results: The quantitative detection results were summarized and compared with the relative copy number results of SMN1/2 and NAIP genes derived from the detection of deletion genotypes by the MLPA method. For the specimens with inconsistent results, the MLPA method was used to recheck the deletion type first to correct the detection results of the MLPA method. After the first round of analysis and correction, for the samples whose test results are still inconsistent, the samples are purified by phenol-chloroform extraction method, numbered blindly, and then re-examined by the new method established in this research, and the re-examination results are analyzed .

3. Test results:

The test results of 500 evaluation samples showed that the 2- ^ΔΔCt values were significantly different between the samples with 1 target gene missing and no deletion, 2 copies of the target gene and 3 copies of the target gene, and the relative copy number analysis results were reliable. The accuracy and sensitivity of molecular diagnosis of deletional SMA reached 100%. This shows that the fluorescent quantitative PCR kit established by the present invention can quickly and easily screen the different copy numbers of SMN1/2 and NAIP genes, and can realize rapid molecular diagnosis of deletion-type SMA, and its sensitivity, accuracy and practical performance meet the requirements of Current mass population screening and routine molecular diagnostic requirements.

The test results are listed in Table 10 and Table 11.

Table 10 Summary of quantitative results of relative copy number of SMN1/2 and NAIP genes in 500 cases of blind analysis of gDNA samples

C_d: deduced gene copy number; C_R: quantitative detection of relative gene copy number; V_R: quantitative detection of 2- ^ΔΔCt ± SD.

Table 11 Summary analysis of diagnostic results of SMA gene deletion in 500 cases of blind analysis of gDNA specimens

Sensitivity=4/(4+0+0)=15/(0+15+0)=481/(0+0+481)=100%;

Specificity=481/(0+0+481)=100%.

Claims (2)

1. a fluorescent quantitative PCR kit for diagnosing human spinal muscular atrophy, the kit includes amplification primers and fluorescent probes, characterized in that, the amplification primers are a pair of specific amplification SMN1 and A common primer for the SMN2 gene, a pair of primers for specifically amplifying the NAIP gene, and a pair of primers for specifically amplifying the reference sequence; the fluorescent probe is a fluorescent probe for specifically detecting the SMN1 gene, a specific detection A fluorescent probe for the SMN2 gene, a fluorescent probe for specifically detecting the NAIP gene, and a fluorescent probe for specifically detecting a reference sequence; wherein, (1) In the shared primers of the pair of specific amplification SMN1 and SMN2 genes, The upstream primer sequence is: 5'-CATCCATATAAAGCTATCTATATATAG-3' (SEQ NO.1), The downstream primer sequence is: 5'-CTTAATTTAAGGAATGTGAGCACCT-3' (SEQ NO.2); (2) In the pair of primers for specifically amplifying the NAIP gene, The upstream primer sequence is: 5'-TTGTGACTTATGAGCCGTACAGC-3' (SEQ NO.3), The downstream primer sequence is: 5'-AGGCTACAGCAGAAGCACTGAAT-3' (SEQ NO.4); (3) In the pair of primers for specifically amplifying the reference sequence, The upstream primer sequence is: 5'-GAACACACATGTCAGAAGTCTAAG-3' (SEQ NO.5), The downstream primer sequence is: 5'-ACCTCCAGTTAGATCTTCACTTCT-3' (SEQ NO.6); (4) the fluorescent probe of the described specificity detection SMN1 gene is: 5'-TTTGATTTTGTCTGAAACCCTGTAAGG-3'(SEQ NO.7), the fluorescein and its quenching group of the probe are 5'-HEX, 3'-BHQ1; (5) the fluorescent probe of described specificity detection SMN2 gene is: 5'-TTTTGATTTTGTCTAAAACCCTGTAAGGA-3'(SEQ NO.8), the fluorescein and its quenching groups of the probe are 5'-FAM, 3'-BHQ1; (6) The fluorescent probe of the specificity detection NAIP gene is: 5'-ATGGATACCACAGGAGATGGCG-3'(SEQ NO.9), the fluorescein and its quenching group of the probe are 5'-CY5,3'-BHQ2; (7) The fluorescent probe of the specific detection reference sequence is: 5'-CAAATGCATCAGATTCCCACAAGCT-3'(SEQ NO.10), the fluorescein and its quenching group of the probe are 5'-ROX, 3'-BHQ2; (8)所述的参比序列为acctccagtt agatcttcac ttctaaagct aagtgagacc tgagacccatgtagacaccc aagaagcttg tggaatctga tgcatttggc ttagacttct gacatgtgtg ttc(SEQ NO.14)，该序列位于NAIP基因端粒侧78kb处的GRCh37/hg19chr5:71107506－71107618区间。 2. a kind of fluorescent quantitative PCR kit for diagnosing human spinal muscular atrophy according to claim 1, is characterized in that, this kit also comprises DNA polymerase, normal genotype control gDNA specimen, magnesium dichloride and dNTPs.