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.
出处
《中华预防医学杂志》
CAS
CSCD
北大核心
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）
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
Vibrio parahaemolyticus
Nucleic acid amplification techniques
Loop-mediatedisothermal amplification
toxR gene
