Relative quantitative detection method of human motor neuron gene copy numbers and kit thereof

Abstract

The invention belongs to the technical field of in-vitro nucleic acid detection and especially relates to a relative quantitative detection method of human motor neuron gene copy numbers and a kit thereof. According to the invention, specific primers and probes to the seventh and eighth exons of SMN1 and SMN2 genes and reference gene RPP40 are respectively designed; at the same time, qualitative detection primers and probes are also designed for three hot spot mutations of Y272C, 11bp-DUP and 4bp-DEL of the SMN1 gene. The kit comprises a container containing detection primer and probe compositions, a reference container and a PCR reaction liquid container. Relative quantitative determinations to the copy numbers of the seventh and eighth exons of SMN1 (in the first and second PCR reactions) and the seventh and eighth exons of SMN2 (in the third and fourth PCR reactions) and qualitative detection to the three hot spot mutations of Y272C, 11bp-DUP and 4bp-DEL (in the fifth PCR reactions) are respectively performed by 5 independent PCR reactions. The kit is strong in specificity, high in sensitivity, convenient, fast and applicable to large-scale popularization and application.

Description

technical field [0001] The invention belongs to the technical field of in vitro detection of human nucleic acid, and in particular relates to a relative quantitative detection method and a detection kit for the gene copy number of human motor neurons. Background technique [0002] The motor neuron survival genes (Survival of Motor Neuron, SMN) located on human chromosome 5 include the motor neuron survival gene 1 (SMN1) at the telomere end and the motor neuron survival gene 2 (SMN2) at the centromere end. Both have 8 exons, namely exon 1, exon 2a, exon 2b and exon 3~8. SMN1 is highly homologous to SMN2, with a difference of only 5 nucleotides (intron 6: -45G→A, exon 7: +6C→T, intron 7: +100A→G and +214A→G, Exon 8: +245G→A). The normal expression of SMN protein is closely related to the function of α-motor neurons in the anterior horn of human spinal cord. When SMN protein is missing or SMN protein is abnormal, it will lead to the degeneration of the alpha motor neurons in...

AI Technical Summary

Problems solved by technology

Large-sample studies have shown that in the carrier population, the SMN2 gene copy number can be as many as 4 or more. For carriers with only 1 copy of SMN1, when SMN1 and SMN2 are amplified with common primers, it will be inevitable. The influence of multi-copy SMN2 gene dominant amplification on single-copy SMN1 gene (because the two PCR primers are the same at this time) will also lead to deviations in relative quantitative results

In addition, these technologies only focus on the deletion mutations of SMN1 exon 7 and exon 8, but fail to detect the Y272C, 11bp-DUP of SMN1 gene exon 6, 4bp-DEL of exon 3, etc. Common hotspot mutations detected

[0006] Therefore, the reliability of the prior art methods cannot meet the needs of the relative quantification of the human motor neuron gene copy number, and there is an urgent need for a reliable SMN1 gene copy number. Relative quantitative detection method for exon 7 and exon 8 copy number of SMN2 gene

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