轮状病毒核酸标准物质研究  

Establishment of a Nucleic Reference Material for Rotavirus Detection

作　　者：徐蕾蕊[1] 魏海燕[1] 马丹[1] 汪琦[1] 张西萌[1] 李丹[1] 付溥博[1] 刘莉[1] 魏咏新[1] 赵晓娟[1] 孙震[1] 曾静[1] Xu Leirui;Wei Haiyan;Ma Dan;Wang Qi;Zhang Ximeng;Li Dan;Fu Pubo;Liu Li;Wei Yongxin;Zhao Xiaojuan;Sun Zhen;Zeng Jing(Inspection and Quarantine Technical Center of Beijing Enter-Exit Inspection and Quarantine Bureau,Beijing 100026)

机构地区：[1]北京出入境检验检疫局检验检疫技术中心,北京100026

出　　处：《中国食品学报》2018年第7期201-209,共9页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家质检总局科技计划项目(2016IK017);"十三五"国家重点研发计划项目(2016YFD0401102)

摘　　要：目的:研制用于食品中轮状病毒聚合酶链式反应检测的核酸标准物质。方法:从RV阳性粪便样品中提取RNA后,利用基因克隆体外转录方法制备RV cRNA纯品,均匀性初检后,稀释至10-4后分装,制备RV核酸标准物质样品。cRNA经核酸测序确定同源性后,采用微滴式数字PCR方法进行均匀性和稳定性研究。RV核酸标准物质的标准值由多家实验室应用ddPCR联合定值获得,并进行不确定度分析。结果:均匀性结果显示RV核酸标准物质组间精密度与组内精密度差异均无统计学意义(P<0.05)。稳定性检验表明室温(20~25℃)3 d,4℃7 d,-20℃21 d及-80℃6个月样品cRNA含量无显著变化。定值研究和不确定度评价得RV核酸标准物质的标准值为6.60×10~7拷贝/μL,其扩展不确定度为0.60×10~7拷贝/μL。结论:RV核酸标准物质均匀性和稳定性符合二级标准物质技术规范,可作为RV PCR检测的标准物质。Objective： To establish a nucleic reference material for rotavirus（RV） PCR detection. Methods： RV RNA was extracted from RV positive stool samples, and then purified RV cRNA were prepared by applying gene cloning and in vitro transcription methods. On the base of preliminary homogeneity test, the purified cRNA were diluted to 10-4 fold and sub packaged with equal volume. Nucleic sequencing was applied to determine the genetic homology of the reference material samples. The homogeneity and stability were tested by droplet digital PCR（dd PCR） method. The standard value of RV reference material were obtained by commissioned laboratories using dd PCR, and the uncertainty was analyzed.Results： There were no significant differences between intraclass variance and interclass variance（P0.05）. Samples were stable at room temperature（20-25 ℃） for 3 days, 4 ℃ for 7 days,-20 ℃ for 21 days, and-80 ℃ for a half year. The RV nucleic reference material samples were valued at（6.60±0.60）×107 copies/μL. Conclusion： The RV nucleic reference material conformed technical specifications of national reference material, and could be used as a reference material for RV PCR testing.

关 键 词：轮状病毒 核酸 数字聚合酶链式反应 标准物质 

分 类 号：R155.5[医药卫生—营养与食品卫生学]