水环境中诺如病毒RT-LAMP快速检测方法的建立及应用  

Establishment and Application of RT-LAMP Rapid Detection Method for Norwalk Virus in Water Environment

作　　者：罗鸿斌[1] LUO Hong-bin(Dongguan University of Technology, Dongguan, Guangdong 52380)

机构地区：[1]东莞理工学院,广东东莞523808

出　　处：《安徽农业科学》2017年第15期57-59,84,共4页Journal of Anhui Agricultural Sciences

基　　金：广东省公益研究与能力建设专项资金项目(2014A020212692);东莞市高等院校;科研机构科技计划一般项目(2014106101006);广东高校省级重点平台和重大科研项目(2016GXJK156)

摘　　要：[目的]基于诺如病毒(NV)基因保守区,设计一套RT-LAMP简并引物,建立能简便、特异、灵敏检测水环境样品中NV的一步法RT-LAMP技术。[方法]通过试验,优化反应温度(64℃)、反应时间(40 min)、Mg2+浓度(2 mmol/L)等参数,建立反应体系。[结果]该方法只需1台水浴锅在1 h内即可完成检测,可直接用肉眼观察结果,检测灵敏度比普通RT-PCR高100倍;简并引物的设计增强了特异性,可检测多个血清型的NV。[结论]在样品检测中,RT-LAMP技术的灵敏度达97.7%,高于RT-PCR的90.7%,更适用于野外样品和简陋实验室的快捷检测。[ Objective] Based on norovirus （NV） gene conserved region, to design a RT-LAMP degenerate primer, and to establish a simple, specific and sensitive method for the determination of NV in water environment samples by RT-LAMP. [ Method ] Through experiments, the reaction system was established by optimizing reaction temperature （64℃ ）, reaction time （40 min） and Mg2- concentration （2 mmol/L）. [ Result] The method only needed a water bath to complete the test in an hour, and could be observed directly with the naked eye, and the sensitivity was 100 times higher than that of the ordinary RT-PCR. The design of degenerate primers enhanced specificity and could detect multiple serotypes of NV. [ Conclusion] In the sample detection, the sensitivity of RT-LAMP technology was 97.7%, higher than 90.7% of RT- PCR. It was more suitable for rapid detection of field samples and crude Laboratorv.

关 键 词：诺如病毒 逆转录 环介导等温扩增 

分 类 号：X832[环境科学与工程—环境工程]