致猪水肿病大肠埃希菌多重PCR检测方法的建立及应用

本研究旨在建立一种快速鉴定致猪水肿病大肠埃希菌的多重PCR检测方法.分别针对大肠埃希菌16S rDNA、志贺毒素Stx2e A亚基和菌毛F18ab A亚基保守序列设计合成3对特异性引物,优化多重PCR反应条件,并进行特异性和敏感性检测.结果显示,阳性对照菌株扩增产物大小分别为1 062、733和313 bp.特异性和灵敏性检测结果表明,与肠炎沙门菌、多杀性巴氏杆菌、胸膜肺炎放线杆菌、副猪嗜血杆菌、支气管败血波氏杆菌和猪链球菌等猪常见致病菌均无交叉反应;菌体直接扩增法最低检出量为1 875 CFU.利用建立的多重PCR检测方法对分离收集的128株大肠埃希菌进行鉴定,得到36株致猪水肿病大肠埃希菌,其中30株既有菌毛F18ab又产志贺毒素Stx2e,另外6株仅产志贺毒素Stx2e.结果表明,本试验所建立的多重PCR检测方法对致猪水肿病大肠埃希菌的快速诊断和流行病学调查具有一定的应用价值.

Establishment and Application of Multiplex PCR to Identify Escherichia coli Causing Swine Edema Disease

The aim of this study was to establish a rapid multiplex PCR detection method for swine edema disease caused by Shiga toxin-producing Escherichia coli (STEC).Three pairs of specific primers based on Escherichia coli 16S rDNA,Shiga toxin Stx2e subunit A and fimbriae F18ab subunit A were designed and the reaction conditions were optimized.At the same time,sensitivity and specificity of multiplex PCR were studied.The results showed that the amplification product sizes of positive control strain were 1 062,733 and 313 bp.The specificity and sensitivity tests showed that there was no cross reaction with Salmonella enteritidis,Pasteurella multocida,Actinobacillus pleuropneumoniae,Haemophilus parasuis,Bordetella bronchiseptica and Streptococcus suis,as little as 1 875 CFU of bacteria could be detected by multiplex PCR.128 strains of pathogenic Escherichia coli isolated from swine were detected by multiplex PCR,36 strains of STEC were found,30 of them contained both Stx2e and F18ab fimbriae genes,and the others only had Stx2e gene.The results showed that the multiplex PCR could be used in rapid diagnosis and epidemiological investigation of swine edema disease.