G6PD缺乏症基因诊断方法的建立

目的：建立有效可行的葡萄糖‐6‐磷酸脱氢酶（glucose‐6‐phosphate dehydrogenase ，G6PD）缺乏症基因突变型检测的经济、快速、无污染的实用方法。方法建立多重 Taqman MGB 探针荧光 PCR体系，对6种国内主要基因突变型（1376G＞ T、1388G＞ A、95A＞G、871G＞A、392G＞ T、1024C＞ T ）进行突变检测，同时加入βactin基因作内参对整个PCR体系进行监控。用30份已知基因型样品进行重复性检测实验。对200例未知样本同时用多重 Taqman MGB探针荧光法和DNA直接测序法进行检测验证。结果建立的多重Taqman MGB探针荧光PCR法同时检测6个国内最常见G6PD基因突变位点，可在1小时内出检测结果，批内与批间的重复性均为100％。200例未知基因型样本检出34例突变，Taqman MGB探针荧光 PCR法分型结果与DNA直接测序法检测结果一致，阳性符合率、阴性符合率均为100％。结论建立的多重Taqman MGB探针荧光PCR法，可在1小时内出结果，PCR后无需开管，是一种快速、有效可行的G6PD缺乏症基因诊断方法。

Development of a new method for genotype of G6PD deficiency

Objective To establish an effective ,feasible ,economic ,rapid and pollution‐free practical assay for genetic diagnosis of glucose‐6‐phosphate dehydrogenase(glucose‐6‐phosphate dehydrogenase ,G6PD)deficiency .Methods A multiple fluorescence PCR system based Taqman MGB probes was performed to amplify the target fragments of G 6PD gene ,and detect the domestic main 6 mutation sites kinds(1376G> T ,1388G>A ,95A>G ,871G>A ,392G> T ,1024C> T) .At the same time ,the beta actin gene moni‐tors the whole PCR system .Repeatability was tested in 30 samples with known genotype .A double‐blind trial for 200 unknown samples was performed by using multiplex fluorescence PCR assay and DNA sequencing simultaneously .Results The established multiple fluorescence PCR assay based Taqman MGB probes was able to detect the most common 6 mutations of G6PD gene simul‐taneously in 1 hour ,and both specificity and accuracy reached 100% .Among the 200 unknown samples ,34 mutational cases was de‐tected .All the genotyping results of multiple fluorescence PCR assay based Taqman MGB probes were in complete concordance with direct DNA sequencing ,and both specificity and accuracy reached 100% .Conclusion The established multiple fluorescence PCR assay based Taqman MGB probes ,detecting samples in 1 hour without open tubes after PCR ,is a rapid ,effective and feasible method of G6PD deficiency gene diagnosis .