Kit for detecting copy number of human motor neuron survival gene SMN1 and analysis method

Abstract

The invention discloses a kit for detecting the copy number of a human motor neuron survival gene SMN1 and an analysis method, an inert multiplex PCR reaction system is established by controlling the use concentration of a core raw material primer in a PCR reaction, on the premise that the use concentration of the primer is limited, the primer is completely consumed after PCR amplification is finished, and the copy number of the human motor neuron survival gene SMN1 is detected. In combination with normalized melting curve analysis of reference genes, precise quantification of SMN1 gene copy numbers of No.7 and No.8 exons is realized; meanwhile, different pathogenic mutants existing in the SMN1 gene are detected through a method of combining ARMS-PCR with a fluorescent dye melting curve, and corresponding gene mutation sites can be realized by analyzing the magnitude of a delta Tm value. The detection method is simple to operate, high in detection accuracy, short in time consumption, low in detection cost and suitable for detection of carriers of the SMA pathogenic gene SMN1 in large-scale population, SMA patients caused by gene mutation can be synchronously achieved while gene copy number variation is detected, the detection coverage is remarkably improved, and missing detection is effectively avoided.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a kit and an analysis method for detecting the copy number of human motor neuron survival gene SMN1. Background technique [0002] Spinal muscular atrophy (SMA) is an autosomal recessive disorder with progressive, symmetrical proximal muscle weakness and muscle atrophy caused by degeneration of motor neurons in the anterior horn cells of the spinal cord. The carrier rate and morbidity rate vary little among different populations. The morbidity rate in newborn infants is about 1 / 10000~1 / 6000, and the carrier frequency is about 1 / 50~1 / 40. According to the age of onset, motor milestones and the degree of disease progression, SMA is divided into five types: type 0, type I (OMIM: 253300), type II (OMIM: 253550), type III (OMIM: 253400) and type IV (OMIM: 271150) ). [0003] The main pathogenic gene of SMA is located in the 5q11.2-13.3 region of chromosome 5, which presents a ...

AI Technical Summary

Problems solved by technology

PCR-RFLP can only be used for qualitative analysis of patients with homozygous deletion of exon 7 of SMN1 gene (that is, 0 copy), and cannot detect carriers of heterozygous deletion type (that is, single copy), and the technology itself also has incomplete enzyme digestion that leads to The results of misjudgment; PCR-DHLPC method and MLPA method can be used to detect homozygous deletion patients and carriers of heterozygous deletion type, and MLPA method can realize 0 copy, single copy, two copies, three copies and four copies of the gene Quantitative analysis, but the operation is complicated, the throughput is low, time-consuming, specific instruments are required, and the price is expensive, so it is difficult to be widely promoted in practical applications

In addition, about 5% of SMA patients or carriers are caused by the presence of SMN1 gene point mutations, and quantitative analysis of gene copy numbers alone cannot effectively diagnose related diseases, resulting in misdiagnosis or missed diagnosis

[0005] In some previous published technologies, the amplification competition mechanism between the target gene and the internal reference gene in the multiple PCR reaction system is constructed by limiting the concentration of dNTPs and / or the concentration of polymerase to a lower level. After the completion of the entire PCR amplification detection, the reaction The consumption of dNTPs in the system is completed, so that the initial concentration of the target gene and the internal reference gene template are consistent with the corresponding proportion relationship between the concentration of the final reaction product after PCR amplification, and a competition in which the double amplification efficiency of the target gene and the internal reference gene tends to be consistent is established. A unique PCR reaction system, thereby realizing the accurate quantification of the SMN1 gene copy number of exons 7 and 8, and overcoming the problems of low detection accuracy, complicated operation, and high detection cost in the prior art

However, due to the difficulty of quantitative detection of dNTPs, in the actual production and quality control process, it is necessary to pay a high cost to detect and control the concentration of dNTPs, which brings a lot of inconvenience to the production and quality control of enterprises

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