水产品和水体中创伤弧菌现场可视化环介导恒温扩增快检方法的建立及应用

Establishment and application of visual loop-mediated isothermal amplification rapid detection method for Vibrio vulnificus in aquatic products and waters

中文关键词:  创伤弧菌  水产品  水体  可视化  环介导恒温扩增技术

英文关键词:Vibrio vulnificus  aquatic products  aquaculture waters  visual  loop-mediated isothermal amplification

中文摘要:

      目的 基于环介导恒温扩增(loop-mediated isothermal amplification, LAMP)技术建立水产品和养殖水域中创伤弧菌的现场可视化快速检测方法。方法 以创伤弧菌作为研究对象, 分别采用煮沸法和离心柱法提取基因组DNA, 优化弧菌DNA提取方法; 以创伤弧菌gyrB基因作为靶基因, 设计筛选最佳的适于LAMP的引物序列, 分别通过LAMP荧光扩增曲线(加SYTO-9荧光染料), 以及LAMP可视化观察扩增产物的颜色变化(加钙黄绿素), 开展特异性、灵敏度和重复性实验分析。结果 煮沸法为适合于弧菌基因组DNA提取的快捷方法; 优化筛选的引物可以特异地检测创伤弧菌, 检测核酸浓度的灵敏度可以达到10 fg/μL, 并且结果稳定、可靠。采用该方法进行实际样品的检测应用, 在558份水体样品和655份水产品样品中, 创伤弧菌阳性检出率分别为9.01%和8.60%, 阳性检出率明显高于传统的分离培养鉴定结果。结论 低技术要求、低设备成本及检测时间短使得可视化LAMP快检方法成为现场可使用的一个理想选择, 对于水产养殖业来说也更方便。

英文摘要:

      Objective To establish a field visual rapid detection method for Vibrio vulnificus in aquatic products and aquaculture waters based on loop-mediated isothermal amplification (LAMP) technology. Methods The genomic DNA of Vibrio vulnificus were extracted by boiling method and centrifugal column method, respectively, to optimize the extraction method of Vibrio DNA; the gyrB gene of Vibrio vulnificus was used as the target gene, and the optimal primer sequence suitable for LAMP was designed and screened, and the color changes of amplified products (plus calcein) were visually observed by LAMP fluorescence amplification curve (plus SYTO-9 fluorescent dye) and LAMP, respectively, to carry out the experimental analysis of specificity, sensitivity and repeatability. Results Boiling method was a quick method for genomic DNA extraction of Vibrio; the optimized primers could specifically detect Vibrio vulnificus with a sensitivity of 10 fg/μL for nucleic acid concentration, and the result was stable and reliable. The method was applied to the detection of actual samples, in 558 water samples and 655 water samples, the positive detection rates of Vibrio vulnificu were 9.01% and 8.60%, respectively, which were significantly higher than the results of traditional isolation and culture identification. Conclusion Low technical requirements, low equipment costs and short detection time make the visual LAMP quick detection method an ideal choice for field use and more convenient for aquaculture.