兔肠致病性大肠杆菌荧光定量PCR检测方法的建立与应用  

Establishment and Application of Fluorescence Quantitative PCR for Enteropathogenic Escherichia coli in Rabbit

作　　者：左海莉 张莹辉[2] 张晓茜 程君生[2] 于建新 彭小薇[2] 董浩 Zuo Haili;Zhang Yinghui;Zhang Xiaoqian;Cheng Junsheng;Yu Jianxin;Peng Xiaowei;Dong Hao(Jiuyuan District Animal Husbandry and Fishery Service Center,Baotou 014060,Inner Mongolia,China;China Institute of Veterinary Drug Control,Beijing 102600,China;National Institutes for Food and Drug Control,Beijing 102629,China)

机构地区：[1]包头市九原区畜牧水产服务中心,内蒙古包头014060 [2]中国兽医药品监察所,北京102600 [3]中国食品药品检定研究院,北京102629

出　　处：《中国动物检疫》2024年第7期81-85,共5页China Animal Health Inspection

摘　　要：为建立一种有效检测肠致病性大肠杆菌(enteropathogenic Escherichia coli,EPEC)的荧光定量PCR检测方法,根据EPEC的eaeA基因序列设计了特异性引物和探针,对引物和探针的最佳浓度分别进行了优化,并研究了其敏感性、特异性和重复性。同时,应用该方法对临床兔粪样品进行了检测,并与国家标准推荐方法进行了比较。结果显示:本研究建立的EPEC荧光定量PCR检测方法检测阳性质粒的最低检测限为2.97拷贝/μL,批内重复和批间重复试验的变异系数均低于3%,临床样品的阳性检出数量高于国标推荐方法。结果表明,该方法特异性良好、敏感性高、重复性理想,能够准确检出兔粪球样本中的EPEC核酸,具有较好的应用前景。In order to develop an effective fluorescent quantitative PCR(qPCR)for detection of enteropathogenic Escherichia coli(EPEC),specific primers and probes were designed based on eaeA gene sequence,and the concentrations of primers and probes were optimized,respectively,followed by evaluation of the sensitivity,specificity and repeatability.Meanwhile,the method was used to detect clinical rabbit fecal samples and compared with the method recommended in national standard.The results showed that the detection limit of the qPCR was 2.97 copies/μL for positive plasmids,the coefficients of variation(CV)of repetition assays in intra-and inter-batch were both lower than 3%,and the number of positive clinical samples detection by qPCR was larger than that by the method recommended in national standard.In conclusion,the established method was with good specificity,high sensitivity and ideal reproducibility,and could be used to accurately detect EPEC nucleic acids in rabbit fecal samples with a good applicationprospect.

关 键 词：肠致病性大肠杆菌 荧光定量PCR eaeA基因 

分 类 号：S851.3[农业科学—预防兽医学]