环介导等温扩增法和PCR法快速检测肠出血性大肠埃希菌O157_H7的比较.docVIP

 环介导等温扩增法和 PCR 法快速检测肠出 血性大肠埃希菌 O157:H7 的比较# 姜容，龙北国，范宏英，曾桂芬，吴娴波** 5 10 15 （南方医科大学公共卫生与热带医学学院微生物学系，广州 510515） 摘要：[目的] 建立基于环介导等温扩增（loop-mediated isothermal amplification，LAMP） 技术的肠出血性大肠埃希菌(Enterohemorrhagic Escherichia coli ,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 101 cfu/mL，PCR 检测限为 102 cfu/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。 关键词：肠出血性大肠埃希菌 O157：H7；环介导等温扩增技术；PCR 技术；rfbE 基因；快 速检测 中图分类号：R378 20 Detection of Enterohemorrhagic Escherichia coli O157：H7 and its comparison by LAMP and PCR JIANG Rong, LONG Beiguo, FAN Hongying, ZENG Guifen, WU Xianbo (School of Public Health and Tropical Medicine, Southern Medical University, 25 30 35 40 GuangZhou 510515) Abstract: [ Objective ] To establish a rapid method of loop-mediated isothermal amplification (LAMP) for detecting Enterohemorrhagic Escherichia coli（EHEC）O157：H7.[ Method ] A set of six primers which specifically to recognize the rfbE gene of EHEC O157:H7 was designed . The LAMP reaction mix was optimized, and the detection sensitivity, specificity and practical samples testing results of LAMP were evaluated by comparing with PCR detection .[ Results ] The LAMP assay correctly identified 7 EHEC O157：H7 strains, but not the 32 non- EHEC O157： H7 strains. The detection limit was 101cfu/ml in pure culture, up to 10-fold more sensitive than that of rfbE-PCR. When applied in food samples, we got the expected result, as well as traditional testing results. [ Conclusion ] The rfbE-based LAMP assay developed in this study was rapid, sensitive, specific and low cost, holding great potential for detection of EHEC O157：H7 in future. Keywords: Enterohemorrhagic Esche