依据CNAS-GL039在Bio-rad CFX96荧光定量PCR仪进行SARS-CoV-2核酸检测的性能验证

The Performance Verification of SARS-CoV-2 on Bio-Rad CFX96 Real-Time PCR Detection System According to CNAS-GL039

目的对本实验室实时荧光聚合酶链反应(RT-PCR)方法检测SARS-CoV-2进行方法学评价,建立本实验室分子诊断定性检验程序性能验证的标准化方法,为日后性能验证工作提供参考。方法依据CNAS-GL039和厂家说明对SARS-CoV-2核酸检测进行符合率、精密度、检测限、交叉反应和抗干扰能力的验证,实验结果在厂家声称的范围内或是满足本实验室的执行标准为性能验证通过。结果国家临检中心室间质评10个质控样本的定性检测结果均符合,符合率验证通过;批内精密度变异系数(CV)为ORF1ab基因:1.11%、N基因:0.95%;批间精密度变异系数(CV)为ORF1ab基因:0.89%、N基因:1.99%、阴性内参:0.54%;变异系数满足厂家声明的≤5%;精密度验证通过;定值质控品S1(2.01×103 copies/mL)稀释至厂家声明的检测限500 copies/mL;靶基因检出率100%,检测限符合要求;病原体交叉反应检测结果均为阴性,干扰物质检测结果均为弱阳性,分析特异性符合要求。结论SARS-CoV-2核酸检测的符合率、精密度、检测限、交叉反应和抗干扰能力的验证,结果均符合检测要求。

Objective To evaluate the performance of a real-time fluorescent polymerase chain reaction(RT-PCR)method for the detection of SARS-CoV-2 nucleic acid in this laboratory and to establish a standardized performance verification method for molecular qualitative diagnostic procedures in laboratory and so as to provide a reference for future performance verifications.Methods The coincidence rate,precision,detection limit,cross-reaction and anti-interference ability of SARS-CoV-2 RT-PCR method were verified according to the recommended method of CNAS-GL039 and manufacturer instructions.When the experimental results were within the range claimed by the manufacturer or met the executive standard of the laboratory,the performance verification was considered as passed.Results The qualitative test results of the 10 quality control samples from the National Center for Interim Quality Evaluation were all in accordance with the results and the coincidence rate passed.The intra-assay precision coefficient of variation(CV)was 1.11%for ORF1ab gene and 0.95%for N gene.The inter-assay precision coefficient of variation(CV)was 0.89%for ORF1ab gene,1.99%for N gene and 0.54%for negative internal control.The coefficient of variation met the manufacturer’s statement,which was≤5%and the precision verification passed.The quality control product S1(2.01×103copies/mL)was diluted to the low limit of detection(500copies/mL)claimed by the manufacturer and the detection rate of target gene was 100%.The detection limit met the requirements.The results of pathogen cross-reactivity test and interfering substances test were all negative and weakly positive,respectively.The analysis specificity met the requirements.Conclusion According to CNAS-GL039,the coincidence rate,precision,detection limit,cross-reaction and anti-interference ability of SARS-CoV-2 RT-PCR method are all verified,and the results meet the detection requirements.