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目的建立一种先天性肾上腺皮质增生症21-羟化酶缺陷的快速基因检测方法。方法选择中国人群21-羟化酶缺乏症相关基因CYP21A2的突变热点Exon3 Del 8bp、Q318X、R356W、IVS2 A/C>G、I172N、P30L和V281L共7个位点进行巢式PCR。设计上述突变热点的单链延伸引物进行扩增,采用SNaPshot技术对上述7个突变热点进行一次性基因分型检测。结果建立了一套满足质量控制标准的CYP21A2基因快速检测的方法,5例阳性干血片标本患者SNaPshot技术和测序技术的结果完全吻合,有2例复合突变,3例纯合突变,显示SNaPshot技术高度的特异性和灵敏度。结论采用SNaPshot技术检测CYP21A2基因具有高通量、准确、对样本容受性高的特点,便于临床应用。
Objective To establish a rapid method for the detection of 21-hydroxylase deficiency in congenital adrenal hyperplasia. Methods Nested PCR was carried out in 7 sites of Exon3 Del 8bp, Q318X, R356W, IVS2 A / C> G, I172N, P30L and V281L mutated in CYP21A2 gene of Chinese 21-hydroxylase deficiency gene. A single-stranded extension primer was designed to amplify the mutant hot spots. SNaPshot technique was used to perform one-time genotyping on the seven mutant hot spots. Results A set of rapid detection method of CYP21A2 gene was established to meet the quality control standards. The results of SNaPshot technique and sequencing technique in 5 positive samples of dry blood slice were in good agreement. There were 2 complex mutations and 3 homozygous mutations, showing SNaPshot technique High specificity and sensitivity. Conclusion The SNaPshot technique has the advantages of high throughput, accurate detection of CYP21A2 gene and its high tolerance to samples for clinical application.