六种新型冠状病毒核酸检测试剂的性能评价及其与核酸提取试剂匹配性分析

Performance evaluation of six coronavirus nucleic acid detection reagents and matching analysis with nucleic acid extraction reagents

目的通过比较6种新型冠状病毒(2019-nCoV)核酸检测试剂的最低检出限、扩增效率、特异性、准确度以及与6种核酸提取试剂匹配性分析,为选择核酸提取试剂、扩增试剂以及临床诊疗提供科学依据。方法将国家标准物质进行浓度梯度稀释得到6个浓度梯度,用广州达安、北京金豪、武汉明德、四川迈克、江苏硕世和上海之江6种2019-nCoV核酸检测试剂(分别简写为DA、JH、MD、MK、SS、ZJ)检测靶基因,评价其最低检出限、扩增效率、准确度、特异性等性能。阳性质控品水平1、2(低中浓度)分别用6种核酸提取试剂提取(编号a~f)搭配6种2019-nCoV核酸检测试剂检测靶基因,比较不同核酸提取试剂和不同核酸检测试剂之间匹配情况,采用双因素方差分析。结果 DA、JH、MD、SS试剂核衣壳蛋白(N)基因最低检出限为7.0×10^(2)拷贝数/ml;JH、MD、SS、MK试剂开放阅读框架1ab(ORF1ab)基因最低检出限为9.39×10^(2)拷贝数/ml;MK、ZJ试剂包膜蛋白(E)基因最低检出限为5.03×10^(2)拷贝数/ml;SS试剂N基因扩增效率89%,ORF1ab基因扩增效率为90%,扩增效率最高。6种2019-nCoV核酸检测试剂特异性均100%;a、b、c、d提取试剂与6种核酸检测试剂匹配性好,但a提取试剂更适合搭配SS、MK和ZJ核酸检测试剂(P<0.05),d提取试剂更适合搭配MD、SS、DA、JH和MK核酸检测试剂(P<0.05),e提取试剂更适合搭配SS核酸检测试剂(P<0.05),f提取试剂更适合搭配DA、JH核酸检测试剂(P<0.05),e和f试剂不适合搭配MK和ZJ。结论 6种核酸检测试剂性能良好,实验室根据实际情况选择合适的提取试剂和检测试剂搭配使用。

Objective To select nucleic acid extraction reagents and amplification reagents by comparing the minimum detection limit,amplification efficiency,specificity,accuracy,and matching analysis with 6 nucleic acid extraction reagents of 6 novel coronavirus(2019-nCoV)nucleic acid detection reagents and provide scientific basis for clinical diagnosis and treatment.Methods The national-standard material of 2019-nCoV nuclear acids was diluted as ths six fradient concentrations.Then,the diluted samples were analyzed by using the six kinds of agents/kits,Guangzhou Daan(DA),Beijing Jinhao(JH),Wuhan Mingde(MD),Sichuan Mike(MK),Jiangsu Shuoshi(SS)and Shanghai Zhijiang(ZJ).The minimum detection limit,amplification efficiency,accuracy,specificity and other properties of these kits were examined.The positive control levels 1,2(low and medium concentration)were extracted by using the six nucleic acid extraction reagents(numbered a-f)with six 2019-nCoV detective kits to detect target genes of 2019-nCoV genome,and compared between different nucleic acid extraction reagents and different nucleic acid detection reagents.Match between the different nucleic acid extraction reagents detection reagents,using two-way analysis of variance.Results The minimum detection limit of Nucleocapsid(N)gene of DA,JH,MD,SS reagent was 7.0×10^(2) copies/ml.The minimum detection limit of open reading frame 1ab(ORF1ab)gene of JH,MD,SS,MK reagent was 9.39×10^(2) copies/ml.The minimum detection limit of envelope(E)gene of MK,ZJ reagent was 5.03×10^(2) copies/ml.SS reagent N gene amplification efficiency was 89%,ORF1ab gene amplification efficiency was 90%,was the highest among six reagents.a,b,c,d extraction reagents and 6 nucleic acid detection reagents match well,But a extraction reagent was more suitable for use with SS,MK and ZJ reagents(P<0.05),d extraction reagent was more suitable for MD,SS,DA,JH and MK nucleic acid detection reagents(P<0.05);e extraction reagent was more suitable for SS nucleic acid detection reagents(P<0.05),f extraction reagent was most suitable for DA,JH Nucleic acid detection reagents(P<0.05);e and f reagents were not suitable for use with MK and ZJ.Conclusion The 6 detection reagents have good performance,and the appropriate extraction reagents and detection reagents should be used in conjunction with the actual conditions of the laboratory.