肠道病毒71型核酸检测试剂国家参考品的建立

Development of national reference panel for detection kit for enterovirus 71 nucleic acids

目的建立肠道病毒71型(enterovirus 71,EV71)核酸检测试剂国家参考品,用于该类试剂的质量评价。方法收集培养多株EV株,包括不同亚型的EV71及不同型别的柯萨奇病毒(Coxsackievirus)株。经测序分析确证,再经商业化核酸检测试剂筛选验证后,分装、冻干组成国家参考品盘。利用数字PCR法(droplet digital PCR,ddPCR)对EV71代表性病毒株C4亚型进行核酸定量,作为最低检测限参考品。采用不同企业的试剂对参考品进行协作标定,分析标定后结果,最终确定参考品的质量标准,并考察其稳定性。结果EV71核酸检测试剂国家参考品由6支阳性参考品、8支阴性参考品、1份精密度参考品和1支检测限参考品组成。其质量标准为:阳性参考品符合率应为6/6;阴性参考品符合率应为8/8;检测限应不高于3.6×10^3 copies/mL;重复检测精密度参考品10次,要求10次结果均为阳性,且对于荧光PCR法,要求其Ct值的CV<5.0%。阳性参考品各样品在室温放置1 d和反复冻融3次条件下相对比较稳定。结论成功建立了评价EV71核酸检测试剂质量的国家参考品。

Objective To develop the national reference panel for detection kit for enterovirus 71(EV71)nucleic acids and apply to evaluate the kits of the same kind.Methods Various EV strains,including EV71 strains of different subtypes and Coxsackievirus strains of different types,were collected and cultured.After identification by sequencing,all the samples were tested and reconfirmed by commercial EV71 nucleic acid detection kits,then were filled and lyophilized to obtain the national reference panel.EV71 with genotype C4,the major circulating strain in China,was quantified by droplet digital PCR and used to evaluate the limit of detection of assays.The requirement for the national reference panel was developed by collaborative calibrations with the reagents from various manufacturers.Meanwhile,the stability of the national reference panel was evaluated.Results The national reference panel for EV71 nucleic acids consisted of six positive samples,eight negative samples and two samples for assessment of precision and the limit of detection respec-tively.The requirement for the national reference panel was as follows:the coincidence rate of positive samples was 6/6;the coincidence rate of positive samples was 8/8;the limit of detection was not more than 3.6×10^3 copies/mL;all the10 repeat test results of the sample for assessment of precision were positive;the coefficient of variation(CV)of Ct value was less than 5.0%(n=10).The positive samples were stable after storage at room temperature for 1 d and after free-zing and thawing for 3 times.Conclusion A national reference panel was successfully developed for assessing the performance of detection kit for EV71 nucleic acids.