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
etection of Enterohemorrhagic Escherichia coli O157：H7 and its comparison by LAMP and PCR
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。
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.
姜容、吴娴波、龙北国、曾桂芬、范宏英
微生物学生物科学研究方法、生物科学研究技术基础医学
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
Enterohemorrhagic Escherichia coli O157：H7loop-mediated isothermal amplificationPCRrfbE generapid detection
姜容,吴娴波,龙北国,曾桂芬,范宏英.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[EB/OL].(2012-05-09)[2025-08-10].http://www.paper.edu.cn/releasepaper/content/201205-124.点此复制
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