检测K88^+肠毒素性大肠杆菌PCR方法的建立

Detection of K88^+ enterotoxigenic Escherichia coli by polymerase chain reaction

以K8 8菌毛结构基因保守序列为靶序列 ,设计合成了一对可扩增长 2 0 1bp的目的片段的引物 ,成功地建立了检测肠毒素性大肠杆菌(ETEC)K88菌毛基因的PCR方法。进行了PCR方法的特异性试验和敏感性试验。对K99+ ,F41 + ,987p+ 参考菌株和鼠伤寒沙门氏杆菌 ,链球菌 ,金黄色葡萄球菌和猪肺疫巴氏杆菌的检测结果均为阴性 ;该检测方法的敏感度可达 1 0个细菌。用此方法对 1 0株腹泻仔猪粪便分离物进行检测 ,结果有 2株阳性 ;与血清学检测的结果一致。结果表明此方法特异性和敏感性都很高 ,可用于临床K88+

K88 fimbriae is one of the adhesive fimbriae antigens of enterotoxigenic Escherichia coli. A PCR method was developed to detect K88 positive Escherichia coli by amplifing K88 fimbriae gene. The primers used in this assay was designed, according to the conserve section of K88 structural gene sequences (K88ab, K88ac, K88ad). The specificity of this PCR method was confirmed by PCR assay to detect ETEC reference strains, Salmonella typhimurium, Streptococcus, Staphylococcus aureus as well as Pasteurella pneumotropica. Its detection limit was 10 CFU target cells per assay. Among 10 Escherichia coli strains isolated from 1 to 7 days old diarrhea piglets, two strains were K88 positive after detected by this PCR method. And the same result was achieved by slide agglutination. The results demonstrated that this PCR was specific and sensitive. It will be useful for identification of K88 positive E. coli strains.