MTHFR 677C／T基因检测室问调查品制备及其应用研究

Development and applications of external quality control materials for MTHFR 677C/T genotypes

目的通过构建体外质粒DNA作为质控样本开展MTHFR677基因检测室间质量评价计划（简称室间质评），评估参评实验室检测能力及存在的问题，提高临床实验室MTHFR基因检测质量。方法利用基因工程技术体外构建含有MTHFR677位点野生型？上下游序列的重组质粒，并采用定点突变技术得到该位点的突变型（T），分别作为野生型和突变型样本，两者等比例混合后作为杂合突变型样本，并制备质控样本盘开展室间质量评价。2016年和2017年室间质评计划为一年两次，样本盘包含5支样本，覆盖MTHFR 677位点各种基因型别。要求参评实验室收到样本后在规定时间内检测样本并网上上报位点基因型检测结果。4次室间质评分别收到26份、28份、52份和56份有效汇报结果。依据回报结果计算各实验室成绩，使用Microsoft Excel软件进行统计分析，汇总各样本的总体符合率，并分析不同检测方法的符合率和检测错误类型。结果体外构建的重组质粒经Sanger测序验证分别含有MTHFR 677位点野生型？和突变型（T）序列，作为质控品在4次室间质评中并无出现检测失败的样本。2016年和2017年4次室间质评中成绩满分的实验室分别为96.15%（25/26），100%（28/28），96.15%（50/52）和98.21%（55/56）。4次室间质评中，样本检测的总体符合率分别为99.23%（129/130），100%（140/140），96.92%（252/260）和98.93%（277/280）。荧光分子杂交和芯片杂交法检测的样本符合率均为100%；荧光PCR法检测的样本符合率为97.5%（39/40），100%（45/45），94.29%（66/70）和100%（95/95）；Sanger测序法样本符合率为100%（20/20），100%（15/15），90%（36/40）和92.5%（37/40）。结论本研究所制备的质控样本能够有效监测试剂检测性能，具有良好的适用性。各实验室在MTHFR 677基因位点检测总体准确率很高，但个别实验室检测能力有待提高。临床实验室的质量控制对于保证检测结果准确性具有十分重要的作用。

ObjectiveTo evaluate the performance of MTHFR 677 genotyping external quality assessment （EQA） program using plasmid DNA constructed in vitro as quality control samples and discuss the problems in clinical laboratories enrolled in the program.MethodsRecombinant plasmid carrying MTHFR 677C locus sequence was constructed as wild type sample and plasmid with MTHFR 677T mutation was generated with site-directed mutagenesis as mutant type sample. Heterozygous mutant samples were obtained after equal proportion of the two plasmids. EQA scheme were held twice a year in 2016 and 2017, and sample panels contained 5 different samples using recombinant plasmid DNA containing all types of MTHFR 677 locus genotypes. Participating laboratories were asked to test samples using their routine methods and report the results before deadlines. 26, 28, 52 and 56 effective reports were received respectively in the four EQA schemes. The scores of each lab were calculated based on their results and the overall compliance of different samples as well as the sensitivity and specificity of different methods were calculated using Microsoft Excel.ResultsMTHFR 677 locus genotypes of the constructed plasmid were verified by Sanger sequencing and there was no failure of sample detection in the four EQA schemes, which suggest that the plasmid has good clinical applicability. About 96.15%（25/26）, 100%（28/28）, 96.15%（50/52）and 98.21%（55/56）of the laboratories submitted correct results for all samples in the four EQA schemes. The overall compliance rate were 99.23% （129/130）, 100%（140/140）, 96.92% （252/260） and 98.93% （277/280） respectively. All laboratories using digital FISH and microarrays got full marks in four EQA schemes. The compliance rates for fluorescent PCR were 97.5% （39/40）, 100% （45/45）, 94.29% （66/70） and 100% （95/95） respectively, while the rates were 100% （20/20）, 100% （15/15）, 90% （36/40） and 92.5% （37/40） for Sanger sequencing.ConclusionsThe recombinant plasmid DNA constructed in this study can effectively detect the performance of reagents with good clinical applicability. The results of EQA programs suggested that the overall accuracy rate of laboratories enrolled was high enough, while some laboratories′ performance still needs to be improved. Quality controls in clinical laboratories were essential to assure the accuracy of results.（Chin J Lab Med, 2018, 41： 749-754）