海产品中创伤弧菌实时浊度LAMP检测方法的建立

Development of a real-time turbidimeter-based loop-mediated isothermal amplification for detection of Vibrio vulnificus in seafood

为建立适合基层实验室快速检测创伤弧菌的环介导等温扩增(LAMP)方法,本研究针对创伤弧菌vvhA基因的保守区域设计4条引物,通过特异性识别vvhA基因中的6个独立区域快速检测创伤弧菌,利用实时浊度仪检测反应过程中产生的白色沉淀,从而实现对LAMP整个反应过程的实时监控。结果表明,本研究建立的创伤弧菌实时浊度LAMP检测方法操作简便、所需时间短、并具有高度特异性,可以在63℃恒温条件下于60 min内完成检测,特异性良好。该方法对培养的创伤弧菌的检测下限为32 cfu。同时,用建立的实时LAMP检测方法对120份海产品进行检测,共检出37份创伤弧菌阳性样品,与传统培养方法结果一致。该方法与传统方法相比,可以节省大量时间,并且对实验仪器和操作人员的要求低,具有良好的实用性。

To establish a rapid method for detection of Vibrio vulnificus, a loop-mediated isothermal amplification （LAMP） assay was developed with a set of primers designed for targeting six distinct sequences of V.vulnificus whA gene, and the real-time monitoring of the LAMP reaction was achieved by real-time ru_rbidimeter, The results showed that the amplification was completed in 1 hour by incubating all of the reagents in a single tube by real-time turbidimeter at 63 ~C. In addition, the specific test demonstrated that the assay was specific to amplify the DNA from V.vulnificus with a detection limit of 32 cfu, but no amplification with the other related bacteria. Furthermore, a total of 120 seafood samples were tested by the established method and the positive samples were 37, which was accordant with the traditional bacteria isolation methods. Compared to the traditional method, this method is time saving, low requirement of instruments and practicability.