水产品中哈维氏弧菌等温快速检测方法优化及比较

Optimization and comparison of isothermal rapid detection methods for Vibrio harveyi in aquatic products

中文关键词:  哈维氏弧菌  荧光LAMP方法  可视化LAMP方法  普通RPA方法

英文关键词:Vibrio harveyi  fluorescence  visual loop-mediated isothermal amplification  recombinase polymerase amplification

中文摘要:

      目的 基于核酸等温扩增技术, 建立荧光法、可视化环介导恒温扩增(loop-mediated isothermal amplification, LAMP)和重组酶聚合酶扩增(recombinase polymerase amplification, RPA)快速检测哈维氏弧菌(Vibrio harveyi)的分析方法, 并对方法进行优化和比较。方法 选择哈维氏弧菌toxR基因为特异靶标序列, 分别设计了2组LAMP引物、3组RPA引物, 对方法进行筛选。优化建立了荧光法LAMP、可视化LAMP、普通RPA 3种等温扩增方法, 对3种方法的特异性和灵敏度进行比较, 并用模拟污染样本验证检测实际样品的灵敏度。结果 基于快速提取的哈维氏弧菌基因组DNA, 本研究所建立的荧光法LAMP、可视化LAMP、普通RPA 3种等温扩增方法均具有良好的特异性, 与同属及近属的细菌没有交叉反应。3种方法的灵敏度分别为5.27 fg/μL (1.99 fg/μL~0.93 pg/μL, 95%置信区间)、0.11 ng/μL、1.07 pg/μL; 荧光LAMP和普通RPA对模拟污染样本的灵敏度分别为7 CFU/mL和97 CFU/mL。结论 本研究建立优化的3种等温快速检测方法结果准确、操作简单, 扩增效率和灵敏度优于文献报道的同类方法, 提高了哈维氏弧菌现场快速检测水平。

英文摘要:

      Objective To establish the analytical methods for rapid detection of Vibrio harveyi by fluorescence, visual loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), based on the isothermal amplification technology of nucleic acid, and optimize and compare the methods. Methods The toxR gene of Vibrio harveyi was selected as the specific target sequence, 2 sets of LAMP primers and 3 groups of RPA primers were designed, the primers were screened, and 3 kinds of isothermal amplification methods of fluorescence LAMP, visual LAMP and ordinary RPA were optimal establishment, the specificity and sensitivity of the 3 kinds of methods were compared, and the sensitivity of the actual sample was verified by simulated contaminated samples. Results Based on the rapidly extracted Vibrio harveyi genomic DNA, the 3 kinds of isothermal amplification methods of fluorescence LAMP, visual LAMP and ordinary RPA established in this paper had good specificity and had no cross-reactivity with bacteria of the same genus and related genus. The sensitivities of fluorescent LAMP, visual LAMP and ordinary RPA were 5.27 fg/μL (1.99 fg/μL-0.93 pg/μL, 95% confidence interval), 0.11 ng/μL, and 1.07 pg/μL, the sensitivities of fluorescent LAMP and ordinary RPA to simulated contaminated samples were 7 CFU/mL and 97 CFU/mL. Conclusion In this paper, the 3 kinds of optimized isothermal rapid detection methods have accurate results, simple operation, amplification efficiency and sensitivity than similar methods reported in literature, and improve the level of on-site rapid detection of Vibrio harveyi.