应用环介导等温扩增技术快速检测副溶血性弧菌  

Rapid detection of Vibrio parahaemolyticus by loop-mediated isothermal amplification

作　　者：吴家林[1] 沙丹[1] 孙燕萍[1] 马广源[1] 张敬平[1] 凌霞[1] 

机构地区：[1]无锡市疾病预防控制中心,江苏无锡214023

出　　处：《中国卫生检验杂志》2013年第6期1481-1484,共4页Chinese Journal of Health Laboratory Technology

基　　金：卫生部科研基金(No.WKJ2006-2-10);无锡市卫生系统"十一五"重大综合发展项目(No.WZD0601);无锡市卫生系统科研项目(No.XM0901)

摘　　要：目的:建立能快速、特异检测副溶血性弧菌的环介导等温扩增技术。方法:针对副溶血性弧菌不耐热溶血素基因(tlh)和热稳定直接溶血素基因(tdh)设计2套引物,建立环介导等温扩增技术检测方法,与PCR法进行比较,并进行了虾肉模拟样品和实际样品检测。结果:环介导等温扩增技术特异性高。灵敏度高于PCR法,对细菌纯培养物的检测灵敏度为101cfu/ml,对虾肉模拟样品检测灵敏度103cfu/g。而PCR法的灵敏度分别为102cfu/ml和105cfu/g。采用环介导等温扩增技术在69份实际样品中检测出11份tlh基因阳性,其中2份tdh毒力基因阳性。结论:初步建立能快速、灵敏、特异地检测副溶血性弧菌的环介导等温扩增技术。Objective： To establish a loop -mediated isothermal amplification （LAMP） technique for rapid detection of Vibrio parahaemolyticus. Methods ： Two sets of primers were designed specifically according to tlh and tdh gene of V. parahaemolyticus. The LAMP system was developed and optimized. The specificity and sensitivity of this system were evaluated by comparing with polymerase chain reaction （PCR）. Artificially - contaminated prawn and practical samples were detected. Results： The LAMP method was highly, sensitive and specific higher than the PCR assay in sensitivity. Sensitivhy of the LAMP detection was 10^1 cfu/ml for bacteria samples and 10^3 cfu/g for V. parahaemolyticus in artificially - contaminated prawn. While those of the PCR assay were 10^2 cfu/ml and 10^5 cfu/g. Eleven V. parahaemlyticus strains were found with tlh primers from 69 practical samples, including toxic bacteria with tdh primers. Conclusion： A rapid, specific and sensitive LAMP technique for detection of V. parahaemolyticus has been studied. The LAMP assay is worthy of popularization, with important significance to disease survey and emergency detection.

关 键 词：环介导等温扩增 副溶血性弧菌 

分 类 号：R446.5[医药卫生—诊断学]