SARS-CoV-2核酸检测试剂盒的制备及样本混检性能分析

Preparation and mixed-samples detection validation of nucleic acid detection kit for SARS-CoV-2

目的开发一种适用于中国大规模人群新型冠状病毒(SARS-CoV-2)感染筛查的核酸检测试剂盒,分析对多样本混合检测的效果,为临床多样本混合检测提供理论指导。方法选用SARS-CoV-2的ORF1ab、N 2个靶基因,并加入RNase P作为内标基因进行试剂盒研发。采用单一变量及正交实验优化试剂盒中3个靶基因引物及探针浓度比例。评价试剂盒的准确性、特异性及灵敏度。收集阳性样本与阴性样本按不同比例混合,采用本试剂盒及市售参比试剂盒分别检测混合样本的SARS-CoV-2,比较不同试剂盒检测不同靶标的检出浓度及扩增Ct值。结果本试剂盒阳性符合率、阴性符合率及特异性均为100%,灵敏度为1×10^(3) copies/mL;不同试剂盒对混合样本中不同靶基因的检出浓度不同,本试剂盒对ORF1ab靶基因的检出限为混合比例1/20,灵敏度高于参比试剂盒(1/10),且检测相同浓度模板的扩增Ct值低于参比试剂盒(P<0.05)。结论本试剂盒ORF1ab靶基因检测灵敏度高,在SARS-CoV-2核酸多样本混合检测中更具优势,适用于大规模人群筛查。

Objective A novel corona virus(SARS-CoV-2)nucleic acid detection kit was developed and performance had been optimized for large-scale population screening in China.The effect of mixed detection of multiple samples was analyzed aiming at providing theoretical guidance for clinical use.Methods The primers and probe sequences for ORF1ab and N announced by China Center for Disease Control and prevention and the primers and probe sequences for RNase P as control gene were synthesized.Single variable optimization and orthogonal experiment were used to get the best concentration ratio of primers and probes for ORF1ab,N and RNase P in fluorescent RT-qPCR detection system.The accuracy,specificity and sensitivity of the kit were investigated.The positive samples and negative samples were collected and mixed according to different proportions,and the SARS-CoV-2 was detected by this kit and the commercial reference kit respectively.The detection concentration for different targetsand the amplification Ct value were compared.Results The positive coincidence rate,negative coincidence rate and specificity of the kit were 100%,respectively。And the sensitivity was 1×10^(3) copies/mL.The detection sensitivity of this kit for ORF1ab target gene was 1/20,higher than that of reference kit(1/10),and the amplification Ct value with same template concentration was lower than that of reference kit(P<0.05).Conclusions This kit has great advantages in screening multi samples detection of new corona virus nucleic acids because of high sensitivity to ORF1ab target gene,and suitable for in large-scale population.