PCR检测肠出血性大肠杆菌O157∶H7

Detection of enterohemorrhagic escherichia coli O157∶H7 by multiplex PCR assay

目的 研究快速、特异、灵敏的检测肠出血性大肠杆菌O15 7∶H7的多重PCR方法 ,并比较单一PCR和多重PCR对检测灵敏度的影响。方法 选择O15 7∶H7的O抗原、H鞭毛抗原及SLT1和SLT2毒素基因特异的 4对引物 ,分别或共同进行PCR扩增 ,检测 4 0株O15 7∶H7和非O15 7∶H7菌株 ,将细菌按 10 1～ 10 6稀释后比较PCR的检测灵敏度。结果 所有O15 7∶H7菌株均在 4 97bp和 6 2 5bp处出现O15 7抗原基因和H7抗原基因产物 ,其产毒株在4 84bp和 (或 ) 2 10bp处出现SLT2和 (或 )SLT1基因产物 ,非O15 7∶H7菌株PCR结果均为阴性 ;单一PCR检测灵敏度为 15 0CFU/PCR反应 ,多重PCR为 >15 0 0CFU/PCR反应。结论 在经过增菌后 ,多重PCR比传统细菌检测方法更特异、快速、灵敏和简便 ,为产毒和非产毒O15 7∶H7的诊断提供了新的手段。

Objective To develop a method of multiplex PCR to detect E.coli O157∶H7 rapidly,specifically and sensitively.To compare its sensitivity with conventional PCR(only one sets of primers).Methods Four pairs of primers were designed from O antigen,H flagellar antigen and Shiga-like Toxin(SLT) 1 and 2 genes.Fourty strains of O157∶H7 and non O157∶H7 were detected by conventional PCR and multiplex PCR amplification.And the sensitivities of PCR were estimated when the strain was diluted from 10 1-10 6.Results O antigen and H flagellar antigen genes amplification generated amplicons of both 497bp and 625bp in all EHEC O157∶H7 strains,SLT1 and(or) SLT2 genes amplification generated amplicons of 210bp and(or) 484bp in toxinogenic O157∶H7.Other non O157∶H7 failed to yield any amplicon under comparable conditions.The sensitivity of detection by conventional PCR was shown to be at least 150CFU per PCR tube and >1 500CFU by multiplex PCR.Conclusion This multiplex PCR method was more specifically,rapidly,efficient after enrichment than traditional bacterial detection.It would become a new method to diagnose the toxinogenic O157∶H7 and nontoxinogenic strain.