Congenital adrenal hyperplasia gene screening kit, screening method and application thereof

Abstract

The present invention discloses a congenital adrenal hyperplasia gene screening kit. The congenital adrenal hyperplasia gene screening kit comprises a PCR amplification primer mixed liquor used for detecting 17 mutation sites including CYP 21A2 gene; an extended primer mixed liquor for the above 17 mutation sites; dNTP; 10*RCR buffer; 25 mM Mg2+ ions; Fast StartTaq enzyme; purification reagent composed of SAP enzyme, Exon I enzyme and a matched buffer; SNaPshot Multiplex mixed liquor; single-site homozygous and heterozygous mutation positive and negative control DNA; and deionized water. According to the technical scheme of the invention, a large number of researches and practices show that, the mutation sites of the congenital adrenal hyperplasia gene in the Chinese population are screened and combined. By utilizing the kit, 17 mutation sites of the specific congenital adrenal hyperplasia gene are simultaneously subjected to multiplex nested PCR amplification, labeling and extending, so that the defects of the existing mutation screening method are overcome. The kit is simple in operation and low in cost. The detection throughput and the accuracy of the detection result are greatly improved compared with the prior art.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a genetic screening kit for congenital adrenal hyperplasia in a Chinese population, a screening method and an application thereof. Background technique [0002] Congenital adrenal hyperplasia (CAH) belongs to a group of common autosomal recessive genetic diseases caused by congenital defects of certain enzymes in the biosynthesis of steroid hormones. The incidence rate of 21-hydroxylase deficiency is 1 / 5000. The clinical phenotype and biochemical manifestations of different types of CAH have their own characteristics based on the type of defective enzyme and the difference in residual enzyme activity. 90%-95% of patients with 21-hydroxylase deficiency have CYP21A2 There are harmful mutations in the gene. The common mutation spectrum and mutation hotspots in the CYP21A2 gene coding region mainly include gene point mutations, small deletions, small insertions, and comp...

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Problems solved by technology

In addition, some mutations did not affect 21-hydroxylase activity and could be considered due to polymorphisms in the 21-hydroxylase genotype

[0004] The main difficulty in molecular level detection is that the true gene CYP21A2 of 21-hydroxylase and the pseudogene CYP21A1P have a high degree of homology, making it difficult to distinguish

The main detection methods for CYP21A2 gene mutations include Southern hybridization, CYP21A2 gene sequencing, multiplex ligation-dependent probe amplification (MLPA), allele-specific polymerase chain reaction (Allele-specific PCR, AS-PCR), denaturing high-performance liquid Denaturing high-performance liquid chromatography (DHPLC), etc., due to the cumbersome, high-cost, low-throughput, or high-quality DNA requirements of the above-mentioned technologies, clinical application is difficult, so it is necessary to create a high-throughput A high-efficiency, low-cost CYP21A2 gene mutation screening method to achieve rapid clinical detection

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