扎伊尔型埃博拉病毒核酸检测试剂国家参考品的研制

Development of national reference panel for Zaire Ebola virus RNA detection kit

目的研制可用于扎伊尔型埃博拉病毒(Ebola virus,EBOV)核酸检测试剂质量评价的国家参考品,并建立相关试剂的质量标准。方法通过分子生物学方法制备含有5种EBOV核蛋白和糖蛋白基因目的片段的RNA噬菌体颗粒、质粒菌液和RNA体外转录产物作为参考品候选样本,使用6种出血热病毒、虫媒病毒培养液和含拉沙热病毒核蛋白和糖蛋白基因的质粒菌液作为阴性参考品候选样本。分别应用培养法和荧光定量PCR确定病毒滴度和核酸浓度,应用RT-PCR、荧光定量PCR对候选样本进行序列鉴定和交叉污染检查。采用RNase A和DNaseⅠ抵抗试验确认RNA噬菌体颗粒的包装完整性。对于序列正确、无污染的候选样本进行稀释分装,发放5家EBOV核酸检测试剂生产企业进行适用性验证,根据结果确定参考品盘的阳性、阴性、最低检测限和精密度组成及试剂质量标准。结果RNA噬菌体颗粒包装的RNA序列与预期完全一致,颗粒包装完整且内含DNA残留污染低于RNA含量的1/10 000,可以用于制备参考品。经适用性验证,确定国家核酸参考品盘包括4份阳性、16份阴性参考品(其中8份型别特异性参考品)、14份最低检测限和2份(4支)精密度参考品,均为冰冻样本。适用性验证结果显示,各申报试剂均能检出扎伊尔型EBOV核酸,但最低检测限存在一定差异。结论建立的扎伊尔型EBOV核酸检测试剂国家参考品适用于现有的5家已申报应急审批的扎伊尔型EBOV核酸检测试剂,可用于相关试剂的质量控制,且对于该类试剂的研发具有重要指导意义。

Objective To develop the national reference panel for Zaire Ebola virus（EBOV） RNA detection kit and establish a quality standard for the relevant kits. Methods The armored RNA（generated from recombinant MS2 bacteriophage）,bacterial material and in vitro transcribed RNA carrying the targeted NP and GP genes of Zaire Ebola virus were used as the candidate samples to establish the positive samples of the panel,while the ones of four non-Zaire Ebola viruses and Lassa virus were used to set the negative samples. In addition,cultures from 6 hemorrhagic fever or arboviruses were also used as negative samples,including Hantavirus,Dengue virus,New Bunya virus,yellow fever virus and Japanese encephalitis virus. The armored RNA was tested by DNase and RNase cleavage. The virus titer and RNA concentration were determined by culture method and fluorescent quantitative PCR respectively. RT-PCR and RT-q PCR were utilized for the sequence validation and contamination check. Then correct samples were subjected to dilutiondispensing and the collaborative study sequentially. Five manufacturers attended the collaborative study to validate the suitability and,based on the result,set up the panel components and reagent quality standard. Results The packed RNA sequence of armored RNA was identical to that of NCBI reference and DNA contaminants were less than 10-4 of the packed RNA. According to the results of collaborative study,the national reference consisted of 4 positive samples,16 negative samples（including 8 type-specific ones）,14 limit of detection（LOD） samples and 2（4 containers） precision samples,all of which were in frozen. Suitability test showed good repeatability but variable LODs between the assays.Conclusion The developed national reference panel was suitable for the Zaire EBOV RNA detection kits which applied for immediate approval,from five manufacturers. The panel may be used for the quality control of the relevant kits,and is of important significance in guiding the development of the kits of same kind.