A群轮状病毒一步法RT-ddPCR方法的建立

Development of a One-Step Reverse Transcriptase Droplet Digital PCR Assay for Detection of Rotavirus Group A Strains

目的:建立A群轮状病毒(rotavirus,RV)一步法反转录微滴式数字聚合酶链式反应(reverse transcriptase droplet digital polymerase chain reaction,RT-ddPCR)方法。方法:筛选特异性引物探针,建立并优化一步法RT-ddPCR方法,确定特异性;制备含A群RV目的片段的RNA标准物质,确定建立方法的检测限、准确度、重复性和复现性。A群RV阳性粪便样品梯度稀释后制作染毒液,制备人工染毒样品,分离病毒与食品基质,浓缩得到病毒悬液,提取RNA后,比较不同食品基质中一步法RT-ddPCR检测结果。结果:建立的一步法RT-ddPCR方法定量限为58000.00~5.80拷贝/μL,具有良好的特异性、准确性、灵敏度、重复性和复现性。各染毒水平下A群RV检出量和回收率在不同食品基质中存在显著性差异(P<0.05)。结论:研究建立的一步法RT-ddPCR方法可准确定量A群RV RNA,食品基质可影响一步法RT-ddPCR检测结果,通过病毒回收率可计算食品中A群RV的实际载量。

Objective:To establish a one-step reverse transcriptase droplet digital polymerase chain reaction(RT-ddPCR)assay for the detection of rotavirus(RV)group A strains.Methods:A set of specific primers and probes was selected for RT-ddPCR.The specificity of this assay was evaluated with RNA samples of other foodborne viruses.A reference material containing the target fragment of RV group A strains was prepared and diluted into several gradients for use in evaluation of the limit of quantitation(LOQ),accuracy,repeatability and reproducibility.Artificially positive samples were prepared by adding the diluted positive fetal sample to different food matrices.Viruses were separated and concentrated from the artificially positive samples,and viral RNA was extracted sufficiently.The effect of food matrices on the quantitative performance of RT-ddPCR was investigated.Results:The RT-ddPCR assay displayed a high accuracy,sensitivity,repeatability and reproducibility.Its LOQ ranged from 58000.00 to 5.80 copies/μL.Statistically significant disparities(P<0.05)in the detectable amount and recovery of virus among different food matrices were discovered at each contamination level.Conclusion:RT-ddPCR could quantitatively and accurately detect RNA of RV group A strains.However,the quantitative results were sufficiently affected by the type of food matrixes.The load of RV group A strains in food could be calculated from the recovery rate.