E.coli O157:H7 LAMP检测方法的建立  

Development of loop-mediated isothermal amplification(LAMP) method for detection of E.coli O157:H7

作　　者：朱海[1] 吕敬章[1] 范放[1] 洪小柳[1] 马淑棉[1] 黄李华[1] 刘慧玲[1] 

机构地区：[1]深圳出入境检验检疫局,广东深圳518067

出　　处：《分子诊断与治疗杂志》2010年第2期98-101,共4页Journal of Molecular Diagnostics and Therapy

基　　金：国家质检总局科技计划项目(2008IK176)

摘　　要：目的建立检测E.coli O157:H7环介导的等温扩增方法(LAMP)。方法选取E.coli O157:H7的rfbE基因设计LAMP引物,优化建立LAMP检测体系。并用该体系和国标法对76份人工污染样品进行了检测。结果所建立的E.coli O157:H7 LAMP体系具有良好的特异性,用该体系对9属13种41株菌进行检测,结果只有E.coli O157:H7的扩增产物电泳结果为阶梯状条带,非E.coli O157:H7均未产生扩增产物,灵敏性为2.7×101CFU/mL。样品及培养基对反应体系没有影响或影响很小。对76份样品进行了检测,E.coli O157:H7 LAMP检测方法与国标法的总符合率为96.1%,2h内即可完成检测。结论本研究建立的E.coli O157:H7的LAMP法不但解决了分子生物学检测对实验室仪器设备的高要求问题,而且检测方法简便、快捷,非常便于基层实验室和现场检测使用。Objective To establish a method of loop-mediated isothermal amplification（LAMP） for detection of E.coli O157：H7. Methods A set of four primers including two outer and two inner primers which specifically to recognize the rfbE gene of E.coli O157：H7 was designed. The LAMP reaction mix was optimized. To compare with standard method （GB/T 4789.36-2008）,76 samples were tested. Results The LAMP assay correctly identified 27 E.coli O157：H7 strains,but 14 non-E.coli O157：H7 strains. Sensitivity of the LAMP assay for direct detection of E.coli O157：H7 in pure cultures and in spiked chicken samples was 2.7×101 CFU/mL. With this method,the detection of E.coli O157：H7 can be finished in less than 2 hours. The coincidence between LAMP and standard method are 96.1%. Conclusion The LAMP assay is a sensitive,rapid and simple tool for the detection of E.coli O157：H7 and will facilitate the surveillance for control of contamination of E.coli O157：H7.

关 键 词：环介导的等温扩增 E.coliO157:H7 rfbE 

分 类 号：R450[医药卫生—治疗学]