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

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

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。

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 rJbE 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 0157：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.