GⅠ和GⅡ诺如病毒RAA可视化快速检测方法的建立  

Establishment of a Visual Recombinase Aided Amplification (RAA) Method for Rapid Detection of GI and GII Noroviruses

作　　者：林志伟[1] 杨艳歌[2] 吴占文 王帅 李涛[2] 李红娜[2] 袁飞[2] Lin Zhiwei;Yang Yange;Wu Zhanwen;Wang Shuai;Li Tao;Li Hongna;Yuan Fei(College of Agriculture,Heilongjiang Bayi Agricultural University,Daqing 163000,Heilongjiang;Key Laboratory of Food Quality and Safety for State Market Regulation,Chinese Academy of Inspection and Quarantine,Beijing 100176;College of Life Science and Biotechnology,Heilongjiang Bayi Agricultural University,Daqing 163000,Heilongjiang)

机构地区：[1]黑龙江八一农垦大学农学院,黑龙江大庆163000 [2]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 [3]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163000

出　　处：《中国食品学报》2023年第12期196-208,共13页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家重点研发计划项目(2022YFF1100900,2022YFF0607900)。

摘　　要：诺如病毒(NoV)是引起全球急性肠胃炎疾病的主要病原体之一,极易爆发传播,增加医疗与经济负担。目的:通过重组酶介导等温扩增技术(RAA)开发一种新型的NoV检测方法。方法:根据ISO TS 15216-2-2013、GB 4789.42规定的GⅠ和GⅡ NoV检测靶标所对应的cDNA序列,设计并筛选最佳RAA引物及探针,确定其对其它常见食源性腹泻病毒的特异性。通过优化确定最短检测时间、反应程序以及反应体系,分析该检测体系下对NoV参考质粒和真实样本检测的灵敏度,从而建立GⅠ和GⅡ NoV RAA可视化快速检测方法。结果:所建立的RAA检测方法特异性好,与其它食源性病毒无交叉反应。在保证扩增效率的前提下,优化后的反应程序可将检测时间缩短为10 min左右,反应成本可减少三分之二。对参考质粒和真实样本检测的灵敏度分别达10^(-2)ng/μL和1 ng/μL。结论:本试验建立的两种NoV检测方法特异性强、灵敏度高,且简单、快速、可视,为今后NoV快速检测奠定良好的基础。Norovirus(NoV)is one of the major pathogens causing acute gastroenteritis diseases worldwide and is highly susceptible to outbreak transmission,increasing medical and economic burden.Objective:To develop a novel detection method for NoV by recombinase aided amplification(RAA).Methods Based on the cDNA sequences corresponding to the GI and GII NoV detection targets specified in ISO TS 15216-2-2013 and GB 4789.42,the optimal RAA primers and probes were designed and screened,and their specificity for other common food-borne diarrhea viruses was determined;the shortest detection time,reaction procedure,and reaction system were determined by optimization,and the analysis of this detection system was performed.The shortest detection time,reaction procedure and reaction system were optimized,and the sensitivity of the assay system for the detection of NoV reference plasmids and real samples was analyzed,thus establishing a rapid visualization method for the detection of GI and GII NoV RAA.The optimized reaction procedure can shorten the detection time to about 10 min and reduce the reaction cost by two-thirds,and the sensitivity of the method can reach 10-2 ng/μL for the reference plasmid and 1 ng/μL for the real sample.The two NoV assays established are specific,sensitive,simple,rapid and visualized,and provide a good basis for future rapid NoV detection.

关 键 词：诺如病毒(NoV) 重组酶介导等温扩增(RAA) 可视化 快速检测 

分 类 号：TS207.4[轻工技术与工程—食品科学]