LAMP技术快速检测沙门氏菌方法的建立及其应用

    田桢干1，阎俊1，陆晔1，王婷2，章琪1，张胜萍2，王传贵2

    1.上海国际旅行卫生保健中心，上海 200135；2.华东师范大学生命医学研究所

    摘要：目的建立和评价一种适合国境口岸及基层检验部门使用的沙门氏菌快速检测方法，在2010年上海世博会保障期间开展应用研究。方法针对沙门氏菌agfA基因，运用环介导等温扩增技术（loop mediated isothermal amplification,LAMP），设计引物进行检测，在65℃恒温条件下、60min特异性扩增，荧光显色观察结果。对建立的方法进行特异性、灵敏度试验及模拟样本灵敏度试验，与荧光定量PCR、胶体金方法进行比对。并对54株临床分离阳性样本加以验证。结果沙门氏菌属6种不同血清型菌株LAMP检测都呈阳性，其他4种肠道细菌均为阴性；LAMP法的灵敏度与RT-PCR方法接近，达103CFU/ml，是胶体金检测法的100~1 000倍；54株沙门氏菌临床分离样本实验符合率达到100%。结论LAMP技术能够在简易的实验条件下快速报告结果，检测成本较低、特异性高、灵敏度高，适合口岸从业人员及基层检验部门进行沙门氏菌的初步筛查工作。

    关键词：沙门氏菌；环介导等温扩增技术；agfA基因；快速筛查

    中图分类号：R378.2　文献标识码：B

    Establishment and application of LAMPtechnology in the rapid

    detection on Salmonella

    TIAN Zhen-gan*,YAN Jun, LU Ye,WANG Ting,ZHANG Qi, ZHANG Sheng-ping, WANG Chuan-gui

    *Shanghai International Travel HealthcareCenter, Shanghai 200135,China

    Abstract:   Objective  To establish and evaluate a rapid method for detecting Salmonellaat ports and basic clinical laboratory.  Methods   Aset of primers were designed to amplify the salmonella agfA geneunder 65℃ for 60 min by LAMP method. The specificity andsensibility of LAMP were evaluated by comparing with RT-PCR andcolloid gold immunity chromatography assay.  Results   All LAMP reactions of 6 serotypes of salmonellawere positive, and others were negative. The detection limit ofLAMP was 103 CFU /ml which was close to RT-PCR and was 100~1000times of colloid gold immunity chromatography assay. The results of54 bacterial strains amplified by LAMP were all positive and thecompliance rate was 100%. Conclusion   The LAMP assayestablished in this study is a sensitive, rapid and simple tool fordetecting Salmonella without expensive apparatus. Thus, it could beused as a primary screen method to detect Salmonella of employeesat ports and basic clinical laboratory.

    Key words:   Salmonella；LAMPTest；agfA gene；Quick detection

    《中国国境卫生检疫杂志》