新型冠状病毒核酸检测的性能验证及评价

Performance verification and evaluation of nucleic acid detection of novel coronavirus

目的验证实时荧光定量聚合酶链反应(RT-qPCR)检测新型冠状病毒(SARS-CoV-2)检测系统的主要性能。方法参考《医学实验室质量和能力的要求》第1部分通用要求GB/T22576.1-2018和第10部分分子生物检验学领域的要求GB/T22576.10-2018对该实验室RT-qPCR检测SARS-CoV-2核酸的检测系统的符合率、重复性、检出限、抗干扰能力、交叉反应等性能特征进行验证及评价。结果该实验室与国家卫生健康委临床检验中心2020年第1、2次SARS-CoV-2室间质评标本的阳性符合率为92%,阴性符合率为100%,总符合率为95%,其中有1例阳性标本未检出阳性,系标本浓度低于最低检测限;弱阳性标本检测的靶基因ORF-1ab和N的Ct值的批内变异系数(CV)分别为1.38%和1.16%,批间CV分别为1.84%和1.72%,均小于5%;检测限浓度(500 copies/mL)标本5次重复检测SARS-CoV-2核酸结果均为阳性;在加入严重急性呼吸综合征冠状病毒(SARS-CoV)、磷酸盐缓冲液(PBS)、人冠状病毒NL63(NL63)、人冠状病毒HKU1(HKU1)、人冠状病毒229E(229E)、中东呼吸综合征冠状病毒(MERS-CoV)、人冠状病毒OC43(OC43)等标本检测中,SARS-CoV-2核酸检测结果均为阴性;在混有血液≤50%、黏蛋白≤0.9 mg/mL等内源性干扰物质的弱阳性标本检测中,SARS-CoV-2核酸检测结果均为阳性。结论RT-qPCR检测SARS-CoV-2核酸的检测系统的各项性能特征与厂家声明相符,可满足预期用途,适用于SARS-CoV-2的临床常规筛查检测。

Objective To verify the main performance of the Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)detection system by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).Methods The coincidence rate,repeatability,sensitivity,detection limit,anti-interference ability and cross-reaction of the RT-qPCR nucleic acid detection system for SARS-CoV-2 was verified and evaluated according to the requirements of Quality and Competence of Medical Laboratories GB/T22576.1-2018 and GB/T22576.10-2018.Results The positive coincidence rate of the laboratory and the Clinical Laboratory Center of National Health Commission for the first and second SARS-CoV-2 laboratory interstitial assessment in 2020 was 92%,the negative coincidence rate was 100%,and the total coincidence rate was 95%.Among them,one positive sample was not detected positive,because the sample concentration was lower than the minimum detection limit.The intra-assay coefficient of variation(CV)of Ct values of target genes ORF-1ab and N detected by weakly positive samples were 1.38%and 1.16%respectively,and inter-assay CV were 1.84%and 1.72%respectively,both were less than 5%.All samples were positive for SARS-CoV-2 nucleic acid after five repeated tests at the limit of detection concentration(500 copies/mL).The results of SARS-CoV-2 nucleic acid test were all negative in sample solution containing SARS-CoV,PBS,NL63,HKU1,229E,MERS-CoV,OC43,and other pathogens.The results of SARS-CoV-2 nucleic acid test were all positive in the weak positive samples mixed with endogenous interfering substances,such as blood≤50%and mucin≤0.9 mg/mL.Conclusion The performance characteristics of the detection system for SARS-CoV-2 nucleic acid by RT-qPCR are in accordance with the manufacturer′s statement and meet the expected use,which is suitable for routine clinical screening and detection of SARS-CoV-2.