副溶血弧菌LAMP检测方法的建立

副溶血弧菌(Vibrio parahaemolyticus)是一种能引起食源性疾病的重要病原菌。首次将一种新颖的核酸扩增技术-环介导等温扩增技术（Loop-Mediated Isothermal Amplification, LAMP）应用于副溶血弧菌的快速检测。针对副溶血弧菌不耐热溶血毒素基因（tlh）设计四条特异性引物（两条内引物和两条外引物）进行LAMP扩增，对扩增反应进行优化，最佳反应时间为60 min，反应温度为60 ℃。对12种细菌共28株菌进行LAMP扩增，仅14株副溶血弧菌得到阳性扩增结果，证明引物具有很高的特异性。副溶血弧菌基因组DNA和纯培养物的检测灵敏度分别约为90 fg和24 cfu/mL。对模拟食品样品进行直接检测，检测限为89 cfu/g。结果表明，该方法检测副溶血弧菌特异性强、灵敏度高，并且操作简便、检测成本低，1 h即可完成，有望发展成为快速检测副溶血弧菌的有效手段。

Development of Loop-Mediated Isothermal Amplification ( LAMP ) Method for Detection of Vibrio parahaemolyticus

Vibrio parahaemolyticus has been considered as one of the most important foodborne bacterial pathogens. In this paper, the loop-mediated isothermal amplification (LAMP) that amplifies DNA with high specificity and rapidity under an isothermal condition was applied for rapid detection of this pathogen for the first time. A set of four primers, two outer and two inner primers, was designed specifically to recognize the thermolabile hemolysin gene (tlh) of V. parahaemolyticus in this study. The LAMP reaction mix was optimized. The most optimal reaction temperature and time of the LAMP assay for the tlh gene were 60 ℃ and 60 min, respectively. Genomic DNAs from 28 bacterial strains including 14 V. parahaemolyticus strains were amplified using LAMP, and no amplicon was observed in other bacterial strains. The detection limit of this LAMP assay was around 90 fg of V.parahaemolyticus genomic DNA and 24 colony forming units for pure cultures. In addition, this method was applied to detect artificially contaminated food samples, and the detection limit was 89 cfu/g for non-cultured artificially contaminated food samples. These results suggested that detection of V. parahaemolyticus by LAMP is an effective and low-cost procedure with high specificity and sensitivity that requires no specialized equipment. This assay is expected to become a valuable tool for rapid detection and identification of V. parahaemolyticus.