猪大肠埃希菌肠毒素基因的多重PCR检测

Detection of Enterotoxin Genes of Escherichia coli from Piglets by Multiplex-PCR Assay

以产肠毒素性大肠埃希菌(Enterotoxigenic Escherichia coli,ETEC)耐热肠毒素(STa、STb)基因和不耐热肠毒素(LT)基因保守序列为靶序列,设计合成了3对可扩增目的片段为182、108和336 bp的引物,建立了检测ETEC耐热肠毒素基因和不耐热肠毒素基因的多重PCR方法.该方法对肠出血性大肠埃希菌(EHEC)EDL933、猪链球菌Streptcoccus、鼠伤寒沙门氏杆菌Salmonella typhimrium和猪肺疫巴氏杆菌Pasteurella pneumotropic的检测结果均为阴性,并且能检测到102cfu/mL稀释度的标准菌.对11株分离自广东佛山地区腹泻仔猪的待检菌株进行检测,结果为:含STa基因的菌株为5株;含STb基因为2株;含LT基因为3株,其中2株同时具有STb和LT基因.结果表明:该方法的特异性和敏感性较高,可用于ETEC的临床快速诊断和流行病学调查.

A method based on the polymerase chain reaction （PCR） was developed to detect heat-stable enterotoxin （STa, STb）genes and heat-labile enterotoxin （LT）gene from enterotoxigenic Escherichia coli （ETEC）. The PCR primers were designed according to the conserved sequence of STa, STb and LT genes and the expected sizes of PCR-amplified productions were 182,108 and 336 bp, respectively. This method was used to detect enterohemorrhagic E. coli （EHEC） EDL933, Streptcoccus, Salmonella typhimrium and Pasteurella pneumotropic strain, none was found to contain STa, STb and LT genes homologous sequences, and standard strains of 10^2 cfu/mL can be detected. Among 11 E. coli strains isolated from piglets with diarrhea which came from Foshan in Guangdong, 5 carried the gene for STa homologous sequences,2 possessed the gene for STb,3 carried the gene for LT,2 of which carried simultaneously the genes for STb and LT. The results confirmed that this PCR method show high specificity and sensitivity for rapid detection of enterotoxin genes and epidemiological investigation of E. coli strains.