多重PCR方法鉴定5类牛源致泻性大肠埃希菌
Development and preliminary application of multiplex PCR method for detection of 5 types of diarrheagenic Escherichia coli
熊昊飞 1张理坤 1程璐 1尚远昊 1冯玉树 1李冰慧 1李勤凡 1张仕强 1王妍1
作者信息
- 1. 西北农林科技大学动物医学学院,陕西杨凌 712100
- 折叠
摘要
为建立鉴定5类牛源致泻性大肠埃希菌(DEC)多重PCR检测方法,对陕西关中奶牛场犊牛粪便中分离的大肠埃希菌进行检测.以 EAEC(astA、aggR、pic)、EHEC(stx1、stx2)、EPEC(escV、bfpB)、EIEC(invE)和 ETEC(lt、stp、sth)5类DEC中11种毒力基因为目标,uidA基因为阳性对照,建立两体系(体系Ⅰ和Ⅱ)多重PCR检测方法,通过改变引物浓度、退火温度等检测条件进行优化,并对其特异性和灵敏度进行检测.结果显示,体系Ⅰ检测EAEC、EHEC,体系Ⅱ检测EPEC、EIEC、ETEC,除astA引物浓度为0.5 μmol/L,其余均为0.3～0.4 μmol/L;退火温度以59 ℃ 最佳;ETEC 的检测下限最低(1.09 × 103 CFU/mL),其余依次为 EHEC(1.27 × 103 CFU/mL)、EIEC(2.43 × 103 CFU/mL)、EPEC(5.71×103 CFU/mL)、EAEC(7.98 × 103 CFU/mL);经验证该方法只对 DEC 毒力基因产生特异性,而对沙门菌、化脓隐秘杆菌、变形杆菌、肺炎克雷伯菌、粪肠球菌、金黄葡萄球菌不产生特异性条带;应用此方法对183株犊牛腹泻源E.coli进行了检测,共检出17株DEC,其中EAEC 6株、ETEC 4株、EPEC 4株、EHEC 2株、EIEC 1株,经单一 PCR验证结果一致.本研究建立的检测5类DEC多重PCR方法可对样品采用两体系、同一 PCR反应程序进行检测.该方法的建立为牛源DEC中常见毒力基因的检测及分子流行病学调查提供了方法与借鉴.
Abstract
This study aims to establish a multiplex PCR method for the detection of 5 common diar-rheagenic Escherichia coli isolated from Guanzhong area of Shaanxi Province,providing reference for the clinical detection of diarrheagenic Escherichia coli.Using 11 virulence genes of 5 diarrhea-genic Escherichia coli as targets and uidA gene as the positive control,a multiplex PCR method was established to detect EAEC.The established multiplex PCR method was optimized by chan-ging primer concentration and annealing temperature,then it was used to detect Escherichia coli i-solated from Guanzhong area of Shaanxi Province,and the results were verified by single PCR method.We established two systems with 11 pathogenic genes,system A was used to detect EAEC,EHEC,system B was used to detect EPEC,EIEC,ETEC.We detected 17 DEC strains,in-cluding 6 EAEC strains,4 ETEC strains,4 EPEC strains,2 EHEC strains and 1 EIEC strains.The results were consistent with the results of the single PCR method.We established a multiplex PCR method using two systems for the detection of five DEC types.It has been proved that this method has high specificity and sensitivity.
关键词
致泻性大肠埃希菌/多重PCR/毒力基因/犊牛腹泻Key words
diarrheagenic Escherichia coli/multiple PCR/virulence genes/calf diarrhea引用本文复制引用
基金项目
陕西省农业科技攻关计划资助项目(2019NY-090)
出版年
2024
NETL
NSTL
万方数据