高效变性液相色谱技术在产前诊断检测母源DNA污染中的应用

摘要

目的 建立一种质控性较高的高效变性液相色谱(denaturing high performance liquid chromatography,DHPLC)技术,检测短串联重复序列(short tandem repeats,STR)位点,用于排除母源污染,提高产前诊断的准确性.方法选取20个处于孕期的患有遗传病的家系,分别提取父母外周血和胎儿羊水DNA,扩增16个STR位点,根据STR数据库报道16个STR位点的等位基因分布范围设定DHPLC 分析片段的大小,进行DHPLC分析.同时,将母体DNA与胎儿羊水DNA以1∶1、1∶4、1∶9、1∶19、1∶99的比例混合,应用DHPLC对vWA STR位点进行检测,确定该技术的检测灵敏度.结果 应用DHPLC 分析16个STR位点,母亲与胎儿等位基因不同的STR位点均在10个以上,排除母源污染的检测率达66.7％,检测灵敏度为1％～10％.结论 DHPLC技术可准确快速地排除羊水的母源污染且具有较高的灵敏度,质控性较好,达到了欧洲要求的检测技术标准,为产前诊断提供可靠的检测质控技术.%Objective To establish a high-quality method for detecting short tandem repeats(STR)using denaturing high performance liquid chromatography (DHPLC) in order to exclude maternal contamination and improve the accuracy of prenatal diagnosis.Methods Two families were recruited.DNA was extracted from blood samples from the parents as well as amniotic fluid.Sixteen STR sites were amplified and analyzed based on the range of allele length reported by a STR database.Ma-ernal DNA was mixed with DNA derived from amniotic fluid samples with the ratio 1 ∶ 1,1 ∶ 4,1 ∶ 9,1 ∶ 19 and 1 ∶ 99.vWA STR site was detected with DHPLC to confirm the sensitivity of detection.Results Sixteen STR sites were analyzed by DHPLC,for which at least 10 were found to be different between the mothers and fetuses.The detection rate,with maternal contamination excluded,was 66.7％.And the sensitivity of detection was 1-10％.Conclusion Maternal contamination of amniotic fluid can be rapidly excluded with accuracy with DHPLC,which features a high sensitivity and good quality control,and can meet the European standards and provide a reliable quality control platform for prenatal diagnosis.