应用环介导等温扩增技术快速检测副溶血弧菌  

Development of a loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus

作　　者：卢昕[1] 王淑京[1] 刘莎[1] 阚飙[1] 逢波[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所,北京102206

出　　处：《中华预防医学杂志》2012年第5期465-467,共3页Chinese Journal of Preventive Medicine

基　　金：日本政府卫生福利部资助项目（H23.Shinkou.shitei-020）;国家科技重大专项（2009ZX10004.101）

摘　　要：目的建立基于环介导等温扩增（LAMP）技术的副溶血弧菌快速检测方法。方法针对副溶血弧菌toxR基因序列设计一套LAMP引物，应用LAMP技术对33株副溶血弧菌、22株其他种属细菌及含不同副溶血弧菌参考菌株（ATCC17802）基因组拷贝数（5×10^0-5×10^5拷贝）／μ1）的样品进行检测，对平行样品分别应用普通PCR法和TaqMan探针实时PCR法进行检测，对3种检测方法的特异度、灵敏度及检测下限及反应时间进行比较。结果LAMP技术、普通PCR法、TaqMan探针实时PCR法检测副溶血弧菌的特异度、灵敏度均为100％（分别为22／22，33／33），检测下限分别为5×10^1、5×10^3、5×10^2拷贝）／μ1，反应所需时长分别为22min、3h、50min。结论与普通PCR、TaqMan探针实时PCR检测相比，LAMP技术检测下限低，检测时长短，适于对副溶血弧菌进行快速检测。Objective This study aimed to establish a loop-mediated isothermal amplification （LAMP） assay for detection of Vibrio parahaemolyticus （ V. parahaemolyticus ）. Methods The specificity of this assay was evaluated by using a panel of 33 strains of V. parahaemolyticus and 22 strains of other species bacteria. The sensitivity was determined by using serial dilutions of V. parahaemolyticus （ ATCC 17802 ） chromosomal DNA （5 ×10^0 -5× 10^5 copies/μ1）. The samples were also tested by using qualification PCR assay and Taqman real-time PCR assay in parallel for comparison with LAMP. Results Both sensitivity and specificity of LAMP assay, PCR assay and Taqman real-time PCR assay were 100% （22/22, 33/33, respectively）. The detection limits of above three methods assay were 5 ^10^1 copies/μ1,5×10^3 copies/μl and 5 ×10^2 copies/μ1, respectively. The reaction period of time needed of the above three assays was 22 rain, 3 h, 50 rain, respectively. Conclusion Compared to qualification PCR assay and Taqman real-time PCR assay, the established LAMP assay was better in low detection limit and less reaction time, which made it an ideal method for quick detection of V. parahaemolyticus.

关 键 词：弧菌 副溶血性 核酸扩增技术 环介导等温扩增 toxR基因 

分 类 号：R440[医药卫生—诊断学]