新型冠状病毒核酸混采对检测结果的影响

Impact of sample pooling strategy on 2019-nCoV RNA detection results

目的评估新型冠状病毒(2019-nCoV)样本混采对提取、扩增步骤的影响。方法取10只阴性咽拭子储存在6 ml病毒保存液中,充分混匀后1∶2、1∶10稀释,加入灭活2019-nCoV病毒培养液,以模拟10混1、5混1和单人份检测。使用a、b、c 3种提取方式和体系不同的核酸提取试剂及A~E 5种模板上样量和检测灵敏度不同的扩增试剂分别进行提取和扩增。结果对于相同的模拟混采样本,a提取的模板比b、c提取的模板检测循环阈值(Ct)分别提前2.10±0.47和3.46±0.62;扩增相同的混采核酸模板,A试剂的N基因Ct值比B试剂提前1.16±0.48,ORF1ab基因Ct提前2.36±0.54。扩增体系中加入10拭子核酸使A试剂检测Ct值滞后1.36±0.32,对B试剂无影响。使用a试剂提取,10混1混采后,C、D、E试剂的扩增Ct值比检测单拭子样本高1.66±0.39。对于400拷贝/ml的10混1样本,C、E试剂均可检出,D试剂的N基因漏检。结论混采引入的大量人基因组DNA干扰提取和扩增效率。在加大样本提取体积、提高提取试剂性能的同时,应选择大模板量上样、检测灵敏度高的扩增试剂,方可提高系统灵敏度和结果稳定性,大大降低漏检风险。

Objective To evaluate the impact of sample pooling strategy on 2019-nCoV RNA detection results.Methods Ten negative swabs were stored in 6 ml virus transport medium,mixed thoroughly and diluted 1∶2 and 1∶10.Inactivated 2019-nCoV culture medium was added to simulate pooling samples:10 pooling samples,5 pooling samples and 1 swab sample.Extraction and amplification were made using three nucleic acid extraction reagents a,b,and c with different extraction methods and systems,as well as five 2019-nCoV detection reagents A-E with various template loading volumes and sensitivities respectively.Results For the same sample,the Ct values of extracted templates a were 2.10±0.47 and 3.46±0.62 earlier than extracted templates b and c.For samples with identical amplifying,the Ct valves of N and ORF1ab gene of A reagent were 1.16±0.48 and 2.36±0.54 earlier than that of reagent B.Adding nucleic acid of 10 negative swabs to the amplification system lagged the Ct values of reagent A by about 1.36±0.32 Ct,while Ct values of reagent B were not affected.Extracted by regent a,a lag of 1.66±0.39 Ct on average was observed in C,D,and E reagents in detecting pooling samples of ten swabs as compared with one swab sample.When extracting 400 copies/ml pooling samples of ten swabs by reagent a,N gene could be detected by reagents C and E,but not by reagent D.Conclusion Large amount of extraneous DNA is introduced by sample pooling,which could interfere the effiency of extraction and amplification.Strategies of using extraction reagents with large loading volume and high effiency,together with amplification reagents with large template volume and low limit of detection are helpful for ensuring detection sensitivity of pooling samples,and greatly reducing the risk of false negative results.