产志贺毒素大肠杆菌的RPA-LF快速检测方法的建立  

Rapid detection of Shiga toxin-producing Escherichia coli with isothermal recombinase polymerase amplification and lateral flow analysis

作　　者：田鑫鑫 郭容 韩先干[1] 王少辉[1] 王权[1] 钟国防[3] 崔立[2] 蒋蔚[1] TIAN Xin-xin;GUO Rong;HAN Xian-gan;WANG Shao-hui;WANG Quan;ZHONG Guo-fang;CUI Li;JIANG Wei(Shanghai Institute of Veterinary Medicine,Chinese Academy of Agricultural Sciences,Shanghai 200241,China;School of Agriculture and Biology,Shanghai Jiaotong University,Shanghai 200240,China;Shanghai Ocean University School of Life Sciences,Shanghai 201306,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]上海交通大学农业与生物学院,上海200240 [3]上海海洋大学水产与生命学院,上海201306

出　　处：《中国兽医科学》2021年第12期1482-1489,共8页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2018YFC1603900);上海市科学技术委员会科研计划项目(21N31901000);兵团财政科技计划项目(2020AB025-03);中央级公益性科研院所基本科研业务费专项资金项目(2020JB07)。

摘　　要：为建立一种快速检测产志贺毒素大肠杆菌的基于侧流层析的重组酶聚合酶扩增技术(RPA-LF)方法,以志贺毒素stx1和stx2基因序列为靶序列,设计了用于RPA-LF方法的特异性引物及探针,并对RPA-LF反应条件进行优化。结果显示,针对stx1和stx2基因的最佳反应温度分别为39℃和37℃,最佳反应时间分别为10 min和15 min。最佳反应条件下,stx1和stx2基因的最低检测限分别为1 pg和10 pg,灵敏度较高。该检测方法能够准确识别含stx1和/或stx2基因的大肠杆菌,且不与其他6种非目的病原菌发生交叉反应,表明本方法特异性良好。本研究建立的针对志贺毒素stx1和stx2基因的RPA-LF快速检测方法灵敏度和特异性较好,且检测时间短,结果直观可视。本试验为产志贺毒素大肠杆菌的筛选诊断和现场的快速检测开辟了一个新的途径。This study was to optimize a recombinase polymerase amplification(RPA)and lateral flow(LF)assay for for rapid detection of Shiga toxin-producing Escherichia coli.The specific primers and probes were designed based on stx1 and stx2 gene sequences,and were used to optimize the optimal temperature and reaction time for RPA reactions.The results showed that the optimal reaction temperatures for stx1 and stx2 were 39℃and 37℃,respectively.The optimal reaction times for stx1 and stx2 were 10 min and 15 min,respectively.Under the optimal reaction conditions,the minimum detection limits of stx1 and stx2 genes were 1 pg and 10 pg,respectively.The RPA-LF assay exhibited good specificity by showing accurately identify for E.coli strains with stx1 and/or stx2 genes but no cross-reaction with 6 non-target bacteria.The RPA-LF rapid detection method for stx1 and stx2 genes established in this study has good sensitivity and specificity.The results were observable within short time by the naked eye.This method opens a good pathway for rapid diagnostic screening and field analysis.

关 键 词：产志贺毒素大肠杆菌 stx1 stx2 重组酶聚合酶扩增技术 侧流层析法 

分 类 号：S852.612[农业科学—基础兽医学]