2019新型冠状病毒核酸qPCR检测失败的原因分析

Causal analysis on the failure of 2019-nCoV nucleic acid qPCR detection

目的探讨本单位开展新冠病毒核酸实时荧光定量聚合酶链反应(qPCR)检测初期,内标扩增阴性和假阳性扩增等扩增检测失败情况的原因。方法对建立新冠病毒核酸检测方法最初两个月的检测数据和实验记录进行回顾性分析,将扩增检测结果(成功或失败)视为二分类变量,结合单因素和多因素Logistic回归分析,研究扩增失败的潜在原因。结果在14983份标本检测中,出现内标扩增阴性229例(1.53%),而假阳性有24例(0.16%)。经分析,发现内标扩增阴性多与仪器设备故障或检测试剂的质量稳定性有关,而假阳性扩增则与全自动核酸提取仪的使用关系密切。结论各级新冠病毒核酸检测人员要严格按照相关指南和专家共识的要求,认真制定标准操作流程和质量管理办法,尤其要加强对仪器设备和检测试剂的可靠性评价工作,确保“检得快、检得出、检得准”。

Objective To explore the reasons for the failure of qPCR detection of 2019-nCoV in the initial stage of method establishment,such as negative amplification of internal control and false positive amplification.Methods The detection data and experimental records of 2019-nCoV nucleic acid detection in the first two months were analyzed retrospectively.The qPCR amplification results(success or failure)were regarded as two categorical variables.Combined with multivariate Logistic regression analysis and constituent ratio analysis,the potential causes of the amplification failure were analyzed.Results Among 14,983 samples,229 samples(about 1.53%)were negative amplification of internal control,while 24 samples(about 0.16%)were false positive amplification.Through statistical analysis,it was found that the negative amplification of internal control was mostly related to the stability of instruments,equipment and detection reagents,while the false positive amplification was closely related to the use of automatic nucleic acid extractor.Conclusion All kinds of 2019-nCoV nucleic acid testing personnel should not only strictly follow the requirements of relevant guidelines and expert consensus,but also conscientiously formulate the standard operating procedures(SOPs)and quality management methods.In particular,we should strengthen the reliability evaluation of instrumentation and testing reagents to ensure the ability of quick,right and accurate detection.