霍乱弧菌环介导等温扩增LAMP技术检测  

Loop mediated isothermal amplification(LAMP) method for detection of Vibrio cholerae

作　　者：匡燕云[1] 叶卫翔[1] 吕敬章[1] 范放[1] 朱海[1] 赵芳[1] 洪小柳[1] 黄李华[1] 

机构地区：[1]深圳出入境检验检疫局食品检测技术中心微生物实验室,深圳518067

出　　处：《中国公共卫生》2009年第11期1310-1312,共3页Chinese Journal of Public Health

基　　金：国家质量监督检验检疫总局科研项目(2008IK162);深圳出入境检验检疫局科研项目(SZ2007011)

摘　　要：目的应用环介导等温扩增技术(LAMP)进行霍乱弧菌的快速检测。方法针对霍乱弧菌的管家基因(mdh)设计4条特异性引物(2条内引物和2条外引物),建立快速检测食品中霍乱弧菌的环介导等温扩增方法;应用该方法对17种细菌共42株菌进行LAMP扩增,并进行确证,全面评估该方法的灵敏度、特异性、准确性等。结果该方法检测21株霍乱弧菌均得到阳性扩增结果,其余21株非霍乱弧菌均未扩增出条带,扩增反应最佳反应时间为60m in,反应温度为65℃。霍乱弧菌基因组DNA和纯培养物的检测灵敏度分别约为40 fg和50 cfu/mL。对模拟食品样品进行直接检测,检测限为70 cfu/g。结论该方法具有特异性强、灵敏度高,操作简便快速、不需要复杂仪器设备。Objective To apply the loop mediated isothermal amplification（LAMP） method for rapid detection of Vibrio cholerae. Methods A set of four primers （ two outer and two inner） was designed specifically to recognize the housekeeping genes（mdh） of Vibrio cholerar. The method of LAMP for rapid detection of Vibrio cholerae in food sample was established. Genomic DNAs from 42 strains of 17 hinds of bacteria were amplified with LAMP. The sensitivity, specificity and precision of the method were evaluated. Results Twenty-one strains of Vibrio cholerae were all found to be positive by the LAMP and no amplicon was observed in other 21 bacterial strains. The most optimal reaction time and temperature of the LAMP assay for the mdh gene were 60 min and 65℃ ,respectively. The detection limit of the LAMP assay was around 40 fg of Vibrio cholerae genomic DNA and 50 colonies forming units for pure cultrues. In addition,the method was applied to detect artificially contaminated food samples, and the detection limit was 70 cfu/g for non-cultured artificially contaminated food samples. Conclusion The results suggest that detection of Vibrio cholerae by LAMP is an effective and low-cost procedure with high specificity and sensitivity that requires no specialized equipment. So it can be extensively used for fast screen of samples.

关 键 词：霍乱弧菌 环介导等温扩增技术(LAMP) 管家基因(mdh) 

分 类 号：R378.3[医药卫生—病原生物学]