星状病毒核酸检测标准物质的研制  

Development of Astrovirus Reference Material Used in Nucleic Acid Amplification Testing

作　　者：徐蕾蕊[1] 魏海燕[1] 林长军[2] 马丹[1] 汪琦[1] 张西萌[1] 李丹[1] 付溥博[1] 刘莉[1] 魏永新[1] 赵晓娟[1] 曾静[1] 

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 [2]辽宁出入境检验检疫局,辽宁大连116001

出　　处：《食品科学》2016年第6期172-177,共6页Food Science

基　　金：国家质检总局科技计划项目(2014IK109);国家重大科学研究计划项目(2011CD932800);质检公益性行业科研专项(201410059;201410080)

摘　　要：目的：研制用于食品中星状病毒核酸检测的c RNA标准物质。方法：利用基因克隆体外转录方法制备星状病毒c RNA纯品,初步定量后稀释至适宜浓度进行分装,其中一部分分装样品添加RNAsafe处理,剩余部分不做处理,制备2种标准物质样品。采用实时荧光定量实时聚合酶链式反应（real-time-polymerase chain reaction,RTPCR）方法检验2种标准物质样品均匀性和稳定性。样品中RNA浓度（拷贝/μL）通过荧光定量RT-PCR和数字PCR联合定值获得,并进行不确定度分析。结果：均匀性结果显示2种样品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20-25℃10 d、4℃14 d、-20℃3个月及-80℃和液氮6个月2种样品c RNA含量无显著变化。RNAsafe（-）标准物质定值为：（1.652±0.143）×10^8拷贝/μL（-80℃）和（1.652±0.135）×10-8拷贝/μL（液氮）。结论：RNAsafe（-）标准物质样品可作为用于星状病毒核酸检测的标准物质。Objective： To develop a reference material for astrovirus used in nucleic acid amplification testing. Methods： Purified astrovirus c RNAs were prepared by applying gene cloning and in vitro transcription methods. On the base of preliminary quantitation, the purified c RNAs were diluted to the appropriate concentration and sub-packaged with equal volume. Two types of astrovirus reference material sample were obtained after some of the sub-packages were treated with RNAsafe, while the rest were not treated. The homogeneity and stability were tested in both types of reference material sample by applying real-time quantitative（RT-q PCR）. The concentrations of astrovirus reference material were determined by combined use of RT-q PCR and digital PC, and the uncertainty was analyzed. Results： There were no significant differences between intra-class variance and inter-class variance（P〉0.05）. Two types of astrovirus reference material were stable at 20–25 ℃ for 10 days, at 4 ℃ for 2 weeks, at-20 ℃ for 3 months, and at-80 ℃ or in liquid nitrogen for half a year. The reference material samples without RNAsafe were valued as（1.652 ± 0.143） ×10^8 copies/μL（preserved at-80 ℃） or（1.652 ± 0.135） ×10^8 copies/μL（preserved in liquid nitrogen）. Conclusion： The samples without RNAsafe could be used as a reference material for astrovirus used in nucleic acid amplification testing.

关 键 词：星状病毒 核酸 定量实时聚合酶链式反应 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]