4种全自动核酸提取系统对新型冠状病毒假病毒提取效果的比较

Comparative evaluation of four automated nucleic acid extraction platforms using 2019-nCoV pseudovirus

目的:应用4种全自动核酸提取仪及其配套试剂提取新型冠状病毒(2019-nCoV)假病毒核酸,并用同1种商品化RT-PCR扩增试剂盒进行检测,探讨检测结果的差异以评估其提取效果,有助于选择合适的核酸提取平台。方法:将2019-nCoV假病毒连续梯度稀释成不同浓度,用4种不同厂家的核酸提取系统(包括全自动核酸提取仪及其配套提取试剂)A、B、C和D提取核酸,再用同1种2019-nCoV核酸检测试剂盒进行检测,比较其检出率和循环阈值(Ct)的差异。结果:在2019-nCoV假病毒原浓度、1:5、1:10和1:100稀释度时,A、B和D提取的核酸中均能100%检测到ORF1ab和N基因,进一步分析发现这3种平台提取的核酸中检测所得的ORF1ab(F=0.061,P=0.941)和N基因(F=0.038,P=0.963) Ct值差异均无统计学意义;但C在2019-nCoV假病毒1:100稀释时所提取的核酸中ORF1ab和N基因的阳性检出率分别为95%和80%。结论:平台C与其他3种平台A、B、D的核酸提取效果间存在差异。核酸提取平台的不同可能影响2019-nCoV核酸检测的灵敏度,并且可能对病毒载量低的样本产生假阴性结果。

Objective: To compare and evaluate four different automated magnetic bead-based nucleic acid extraction instruments and paired kits for purifying nucleic acids from 2019-nCoV pseudovirus. Methods: Nucleic acids were extracted from serial dilutions of 2019-nCoV pseudovirus using four nucleic acid extraction platforms A, B, C and D, and detected by the commercial 2019-nCoV nucleic acid detection kit. Results: Both ORF1 ab and N genes were detected in the nucleic acids extracted from 2019-nCoV pseudovirus and serial dilutions including 1:5, 1:10 and 1:100 by A, B and D, and there was no significant difference between cycle threshold(Ct) values of ORF1 ab(F=0.061, P=0.941) and N genes(F=0.038, P=0.963) detected by the same amplification kit in the nucleic acids extracted by A, B and D;but the positive detection rates of ORF1 ab and N genes in the nucleic acids extracted from 1:100 dilution of 2019-nCoV pseudovirus using C were 95% and 80%, respectively. Conclusion: The platform C was different from the other three platforms A, B and D in the nucleic acid extraction. The nucleic acid extraction platform may influence the sensitivity of detecting 2019-nCoV and have the potential to generate false-negative results especially in the samples with low viral load.