Rapid,specific and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification targeted to vvhA gene

Vibrio vulnificus is an estuarine bacterial pathogen for human.The rapid,specific and sensitive detection of V.vulnificus is urgently needed for early disease diagnosis and timely treatment of V.vulnificus infection.In the study,a loop-mediated isothermal amplification（LAMP） technique was developed for V.vulnificus detection with a set of primers,composed of two out primers and two inner primers targeted to vvh A gene.The optimal amplification temperature was 63°C and the reaction only took 35 min.The amplification products could not only be detected by agarose gel electrophoresis with ladder-like pattern bands,but also could be visualized using calcein with naked eye directly.Forty-five strains were tested for the specificity of LAMP assay,and all the V.vulnificus strains were identified correctly while other strains were negative results.The sensitive of the new LAMP assay was 100-fold more sensitive than the conventional PCR.Meanwhile,all the V.vulnificus strains were detected correctly in spiked,clinical and environmental samples by the new LAMP assay.Compared with other well-known techniques,the new LAMP assay targeted to vvh A gene was extremely rapid,simple,sensitive and specific for V.vulnificus identification.

环介导等温扩增技术检测创伤弧菌方法的建立与应用

根据创伤弧菌vvhA基因设计4条特异性引物,采用环介导等温扩增方法(LAMP)建立创伤弧菌快速检测方法。结果表明,建立的LAMP方法具有高度特异性,经对13株细菌进行扩增,创伤弧菌为LAMP阳性,其他菌株均为LAMP阴性。该方法对培养的创伤弧菌的检测下限为51CFU/mL,可在60min内完成扩增反应。用建立的LAMP方法对120份海产品进行检测,共检出37份LAMP阳性样本,与传统培养方法结果一致。该方法与传统方法相比,可节省大量时间,且对实验仪器和操作人员的要求低,具有良好的实用性。

海产品中创伤弧菌实时浊度LAMP检测方法的建立

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。