核酸-免疫层析法快速检测肠道侵袭性大肠埃希氏菌  

Rapid detection of Enteroinvasive Escherichia coli by nucleic acid immunochromatography

作　　者：赵雪龙 夏伟诚 刘道峰[3] 刘成伟[3] 山珊 赖卫华[1] ZHAO Xuelong;XIA Weicheng;LIU Daofeng;LIU Chengwei;SHAN Shan;LAI Weihua(State Key Laboratory of Food Science and Technology,Nanchang University,Jiangxi Nanchang 330047,China;Nanchang Key Laboratory of Microbial Resources Exploitation&Utilization from Poyang Lake Wetland,College of Life Sciences,Jiangxi Normal University,Jiangxi Nanchang 330022,China;Jiangxi Province Centre for Disease Control and Prevention Jiangxi Province Key Laboratory of Diagnosing and Tracing of Foodborne Disease,Jiangxi Nanchang 330029,China)

机构地区：[1]南昌大学食品科学与技术国家重点实验室,江西南昌330047 [2]江西师范大学生命科学学院南昌市鄱阳湖湿地微生物资源开发与利用重点实验室,江西南昌330022 [3]江西省疾病预防控制中心江西省食源性疾病诊断溯源重点实验室,江西南昌330029

出　　处：《中国食品卫生杂志》2022年第3期524-530,共7页Chinese Journal of Food Hygiene

基　　金：国家自然科学基金(82003467);江西省重点研发计划(20192BBGL70053,20192BBG70069);江西省自然科学基金面上项目(2020BAB206066);江西省卫健委科技计划(202130977)。

摘　　要：目的建立快速检测肠道侵袭性大肠埃希氏菌(EIEC)的核酸-免疫层析方法。方法利用不对称聚合酶链式反应技术制备目标单链DNA,结合免疫层析技术分型检测EIEC的毒力基因invE和大肠埃希氏菌标志基因uidA。结果在不对称PCR体系中,uidA和invE最佳上下游引物比例为1∶3,最佳引物浓度(下游)分别为0.2μmol/L和0.25μmol/L,最佳扩增循环次数为40。本方法最低检测限为3.97×10^(-3)ng/μL的基因组DNA,特异性与PCR-凝胶电泳法相当。结论本方法具有操作简便、快速、成本低、检测结果准确以及特异性良好等优点,可分型检测EIEC、非EIEC大肠埃希氏菌和非大肠埃希氏菌,适用于基层实验室使用。Objective To establish a nucleic acid immunochromatography method for rapid detection of Enteroinvasive Escherichia coli(EIEC).Methods The target single-stranded DNA was prepared by asymmetric PCR,the invE which was the virulence gene of EIEC and uidA which was the marker gene of Escherichia coli were detected by immunochromatography.Results In the asymmetric PCR system,the optimal ratio of forward and reverse primer for uidA and invE was 1∶3,and the optimal primer concentrations(reverse primer)were 0.2μmol/L and 0.25μmol/L,respectively.The optimal cycle number of amplification was 40.The lowest detection limit of this method was 3.97×10^(-3)ng/μL,and the specificity was comparable to that of PCR-gel electrophoresis.Conclusion The method which had the advantages of convenience,less time consuming,low detection cost,good accuracy and specificity,was suitable for primary laboratory.

关 键 词：检测 肠道侵袭性大肠埃希氏菌 毒力基因 免疫层析 

分 类 号：R155[医药卫生—营养与食品卫生学]