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( ATCC 17802)基因组拷贝数(5×100 ～5×105拷贝/μl)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5×101、5×103、5×102拷贝/μl,反应所需时长分别为22 main、3h、50 min.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.

abstractsObjective This study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for detection of Vibrio parahaenolyticus( V.parahaemolyticus ).Methods The specificity of this assay was evaluated by using a panel of 33 strains of V.parahaemolyticcus and 22 strains of other species bacteria.The sensitivity was determined by using serial dilutions of V.parahaemolyticas ( ATCC 17802 ) chromosomal DNA (5 × 100 -5 × 105 copies/μl).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 × 101 copies/μl,5 × 103 copies/μl and 5 × 102 copies/μl,respectively.The reaction period of time needed of the above three assays was 22 min,3 h,50 main,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.