检测肠出血性大肠埃希菌O157:H7的环介导等温扩增和PCR法比较

目的建立基于环介导等温扩增(LAMP)技术的肠出血性大肠埃希菌(EHEC)O157:H7快速简便检测方法,并与PCR进行比较。方法针对EHEC O157:H7保守的rfbE基因序列,设计特异性引物,建立LAMP和PCR检测技术并优化其反应条件,比较这两种检测方法的灵敏度、特异性和对实际样品的检测结果。结果成功建立了LAMP和PCR检测体系,灵敏度检测结果显示,LAMP检测限低至10 cfu/ml,PCR检测限为102cfu/ml,LAMP检测灵敏度高于PCR;对39株近源菌进行检测,结果显示,LAMP法仅7株EHEC O157:H7得到阳性结果,非EHEC O157:H7菌株均为阴性,PCR产物则出现非特异性条带,LAMP法特异性比PCR法高。对于32份猪肉样品的检测,LAMP和PCR与常规检测结果一致,3份样品检测阳性。结论 LAMP检测技术的特异性和灵敏度较PCR高,并且操作简便、检测成本低,耗时短,更有望发展成为快速准确检测EHEC O157:H7的有效手段。

Loop-mediated isothermal amplification for detecting enterohemorrhagic Escherichia coli O157:H7: a comparison with PCR

Objective To establish a rapid method of loop-mediated isothermal amplification(LAMP) for detecting enterohemorrhagic Escherichia coli(EHEC) O157:H7.Methods Six primers that specifically recognized the rfbE gene of EHEC O157:H7 were designed.Under the optimized reaction conditions,LAMP and PCR were evaluated for the sensitivity and specificity in the detection of 39 laboratory samples of EHEC O157:H7 strains,and their detection results of contaminated fresh pork samples were compared.Results LAMP assay correctly identified all the 7 EHEC O157:H7 strains and showed negative results for all the 32 non-EHEC O157:H7 strains.The detection limit of LAMP was much lower than that of rfbE-PCR(10 vs 100 cfu/ml).In the detection of the contaminated pork samples,both LAMP and PCR yielded results consistent with those by the conventional detection method.Conclusion The rfbE-based LAMP assay can serve as a rapid,sensitive,specific and low-cost means for detecting EHEC O157:H7 strain.