通用引物检测志贺毒素和紧密素多个基因亚型  

Universal primers detection of Shiga toxin and intimin variants

作　　者：张雪寒[1] 汪伟[1] 何孔旺[1] 倪艳秀[1] 温立斌[1] 李彬[1] 王小敏[1] 周俊明[1] 

机构地区：[1]江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室/国家兽用生物制品工程技术研究中心,南京210014

出　　处：《中国人兽共患病学报》2014年第6期607-611,617,共6页Chinese Journal of Zoonoses

基　　金：江苏省支撑计划(No.BE2011771);国家自然科学基金(No.31201919)联合资助~~

摘　　要：目的旨在建立同时检测stx2和intimin两个基因多个变型的双重PCR快速检测技术，更好满足实验室和临床检测的需求。方法以stx2主要的6个亚型和intimin主要的27个基因亚型为靶序列，设计引物，建立双重PCR方法，分析其敏感性、特异性，并检测模拟制备的阳性样品。结果stx2-intimin双重PCR扩增片段分别为388bp和609bp，检测大肠杆菌纯培养物最低限介于10～100cFu／mL，增菌样品最低检测限介于1～10CFU／g，特异性检测intimin和stx2的多个亚型。结论Stx2-Intimin二重PCR技术灵敏、特异、通用和高效。Shiga toxin and intimin are the most important pathogenic E. coli virulent factors. In this study, we aimed to develop a method to simultaneously detect multiple shiga toxin 2 （stx2） and intimin variants in order to satisfy clinical and laboratory tests. Based on the 6 variants gene of stx 2 and 27 variants gene of intimin, primers were designed to analyze their usability by testing their sensitivity and specificity to develop a duplex PCR. It was further assessed by detecting artificially contaminated samples. The amplicon size of stx2 and intimin was 388 bp and 609 bp, respectively. The lowest detection limit of stx2-intimin duplex PCR ranged from 10 to 100 CFU/mL for E. coli culture, 1 to 10 CFU/g for the enriched contaminated samples. Target genes were amplified from human pathogenic E. coli but not from Salmonella, E. coli K88, E. coli K99, E. coli 987 p, E. coli F41, Shigella dysenteriae, Shigella flexneri type 2, or E. coli K12. Stx2-intimin duplex PCR could specifically detect both shiga toxin 2 variants and eae variants from contaminated samples and it is a sensitive, specific, univer- sal and effective technique.

关 键 词：大肠杆菌致病群 志贺毒素 紧密素 变异型 PCR 

分 类 号：R378.21[医药卫生—病原生物学]