双重荧光RT-ERA技术快速检测贝类食品中诺如病毒  

Rapid Detection of Norovirus in Shellfish by Dual Fluorescence Reverse Transcription-Enzymatic Recombinase Amplification

作　　者：吴占文 王帅 康婕 李涛[1] 李红娜[1] 蔡杰 张昊 杨艳歌[1] 袁飞[1] WU Zhanwen;WANG Shuai;KANG Jie;LI Tao;LI Hongna;CAI Jie;ZHANG Hao;YANG Yange;YUAN Fei(Key Laboratory of Food Quality and Safety for State Market Regulation,Chinese Academy of Inspection and Quarantine,Beijing 100176,China;College of Life Science and Biotechnology,Heilongjiang Bayi Agricultural University,Daqing 163000,China;Shanxi Provincial Institute of Product Quality Supervision and Inspection,Xi’an 710048,China)

机构地区：[1]中国检验检疫科学研究院,国家市场监管重点实验室(食品质量与安全),北京100176 [2]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163000 [3]陕西省产品质量监督检验研究院,陕西西安710048

出　　处：《食品科学》2023年第18期355-363,共9页Food Science

基　　金：中国检验检疫科学研究院基本科研业务费专项(2022JK36);“十三五”国家重点研发计划重点专项(2018YFC1603606)。

摘　　要：目的:基于逆转录-酶促重组等温扩增(reverse transcription-enzymatic recombinase amplification,RTERA)技术,以MS2作为模式过程控制病毒,建立一种贝类食品中GI与GII型诺如病毒(norovirus,NoV)双重荧光RT-ERA快速检测方法。方法:分别将GI、GII NoV参考靶序列克隆到带有T7启动子的载体中,通过体外转录的方式获得高纯度NoV RNA参考样品。以设计的MS2噬菌体引物探针,与实验室前期筛选的GI、GII NoV引物探针进行双重荧光RT-ERA实验,并对反应体系和反应程序进行优化,分析方法的灵敏度。最后,采用建立的方法开展真实样本检测,以GB 4789.42-2016《食品微生物学检验诺如病毒检验》为参比方法进行检测结果对比,确定方法的准确性和可行性。结果:建立MS2与GI、MS2与GII两组双重荧光RT-ERA检测方法,最佳引物及探针体积分别为4.1μL与1.8μL,检测时间可缩短至10 min左右,且最低可检出102拷贝/μL的NoV核酸样品。应用所建立的方法对29份真实样品进行检测,检测结果与GB 4789.42-2016一致。结论:本研究建立的双重荧光RT-ERA检测方法可实现对MS2、GI和GII NoV的同时检测,且灵敏度高、准确性强,为今后NoV现场快速检测提供一定基础。Objective:To establish a dual fluorescence reverse transcription-enzymatic recombinase amplification(RT-ERA)method for the rapid detection of GI and GII noroviruses(NoV)in shellfish using MS2 as the model process control virus.Methods:The target sequences of GI and GII NoV were individually cloned into vectors with a T7 promoter.Then,high-purity RNA was obtained by in vitro transcription.Dual fluorescence RT-ERA was performed using the primers and probes of MS2 phage designed in this study and the primers and probes of GI and GII NoV selected previously.The reaction procedure and system were optimized.The sensitivity of the method was analyzed.Finally,this method was used to detect real samples,and its accuracy and viability were determined by comparing the results with those obtained by the reference method specified in the national standard GB 4789.42-2016.Results:The optimal volumes of primers and probes for the dual fluorescence RT-ERA assay were 4.1 and 1.8μL,respectively.The method took only about 10 min to perform,and could detect as low as 102 copies of NoV nucleic acid.When the established method was applied to detect 29 real samples,the results were consistent with those from GB 4789.42-2016.Conclusion:The dual fluorescence RT-ERA assay can simultaneously detect MS2,GI and GII NoV with high sensitivity and accuracy,which will lay a foundation for rapid on-site detection of NoV in the future.

关 键 词：诺如病毒 逆转录-酶促重组等温扩增技术 双重荧光 快速检测 贝类 

分 类 号：TS201.6[轻工技术与工程—食品科学]