大肠埃希菌O157:H7分离鉴定及毒素基因多重PCR检测

Identification of O157:H7 of Escherichia col and detection of toxin gene by multiple PCR

目的了解大肠埃希菌O157:H7(EHEC O157:H7)在云南战区感染性腹泻患者及猪粪便中检出情况。方法将标本接种mEC增菌液中增菌,免疫磁珠磁珠富集后,分别接种科玛嘉显O157显色琼脂和山梨醇麦康凯(S-MAC)琼脂平板培养,MUG初筛,肠杆菌科细菌生化鉴定编码系列(API-20E)和VITEK32全自动微生物生化分析仪鉴定。应用多重PCR扩增检测志贺毒素1、2(SLT1/SLT2)和侵袭相关基因。小白鼠腹腔注射法测定其毒力。K-B法做抗生素药敏试验。结果从腹泻患者和猪中均检出EHEC O157:H7,具有志贺毒素SLT1/SLT2和侵袭相关基因的三个毒素基因。毒力试验使小白鼠发病死亡。药敏试验对14种抗生素敏感。结论云南战区存在EHEC O157:H7感染。建立和优化的多重PCR方法,能快速、敏感特异的检测EHEC O157:H7毒素基因,为重大食源性疾病处理、诊治及流行病学调查提供了强有力的技术手段。

Objective To identify the the exixtence of O157：H7 of Escherichia col in infective diarrheal patients in Yunan area and detect the toxin gene and drug resistance of enteric hemorrhagic E.coli（EHEC）.Methods The specimens were enriched in enrichment culture and noculated with CHROM agar medium and orbital macConkey agar after enriched with immunomagnetic beads,screened with MUG,then identified by biochemical identification coding system（API20E）and VTTEK32 automatic microological biochemical analyzer.Three toxic genes including Shigella toxinI and II（SLT1/ SLT2）amd invasive relevant genes could be simultaneously detected after amplified by multiple PCR.The virulence was determined in mice by peritoneal injection.While the drug sensitivity test was completed using K-B method.Results EHEC 0157 H7 possessing three toxic genes were detected from diarrheal patients and pigs.The mice died of virulence test.The results of drug sensitivity test showed that the pathogens were sensitive for 14 antibiotics.Conclusion There were EHECO157H7 and multiple PCR allowed raid specific and sensitive detection of toxic genes of EHECO157H7 to have offered a powerful technique.