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

作　　者：熊昊飞 张理坤 程璐 尚远昊 冯玉树 李冰慧 李勤凡[1] 张仕强[1] 王妍[1] XIONG Haofei;ZHANG Likun;CHENG Lu;SHANG Yuanhao;FENG Yushu;LI Binghui;LI Qinfan;ZHANG Shiqiang;WANG Yan(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学动物医学学院,陕西杨凌712100

出　　处：《中国兽医学报》2024年第1期107-112,120,共7页Chinese Journal of Veterinary Science

基　　金：陕西省农业科技攻关计划资助项目(2019NY-090)。

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0.5μmol/L,其余均为0.3~0.4μmol/L;退火温度以59℃最佳;ETEC的检测下限最低(1.09×10^(3)CFU/mL),其余依次为EHEC(1.27×10^(3) CFU/mL)、EIEC(2.43×10^(3) CFU/mL)、EPEC(5.71×10^(3) CFU/mL)、EAEC(7.98×10^(3) 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。This study aims to establish a multiplex PCR method for the detection of 5 common diarrheagenic 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 diarrheagenic 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 changing primer concentration and annealing temperature,then it was used to detect Escherichia coli isolated 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,including 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 

分 类 号：S852.61[农业科学—基础兽医学]