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Detection of Vibrio anguillarum and Vibrio alginolyticus by Singleplex and Duplex Loop-Mediated Isothermal Amplification (LAMP) Assays Targeted to groEL and fklB Genes.
International Microbiology ( IF 3.1 ) Pub Date : 2019-05-16 , DOI: 10.1007/s10123-019-00079-z
Mahbubul Pratik Siddique 1, 2 , Won Je Jang 1 , Jong Min Lee 3 , Md Tawheed Hasan 1, 4 , Chang-Hoon Kim 5 , In-Soo Kong 1
Affiliation  

Singleplex and duplex loop-mediated isothermal amplification (LAMP) assays were developed for detecting Vibrio anguillarum, a major bacterial pathogen of fish, and Vibrio alginolyticus, a pathogen of fish and humans, separately and simultaneously from contaminated seawater by targeting the groEL gene of V. anguillarum, which encodes a molecular chaperone protein, and the fklB gene of V. alginolyticus, which encodes a 22 kilodalton (kDa) peptidyl prolyl isomerase. The optimal reaction conditions to produce consistent results were 65 °C for 30 min, 63 °C for 30 min, and 63 °C for 40 min for the groEL (singleplex for V. anguillarum), fklB (singleplex for V. alginolyticus), and groEL + flkB (duplex) LAMP assays, respectively, analyzed via visual detection methods (use of calcein, and SYBR Green I) and agarose gel electrophoresis. The assays were found to be species-specific, as closely related Vibrio spp. were not detected. The limits of detection (LoDs) of the LAMP assays for DNA template from pure culture and artificially contaminated seawater were 10 and 14 fg (groEL assay; for V. anguillarum), 12.5 and 17 fg (fklB assay; for V. alginolyticus), and 50 and 70 fg (duplex assay) per reaction, respectively, which were much better than the LoDs of conventional polymerase chain reaction (PCR). Singleplex and duplex LAMP assays were found to be rapid, species-specific, and sensitive for the detection of V. anguillarum and V. alginolyticus and are applicable to laboratory and field diagnostics.

中文翻译:

通过针对groEL和fklB基因的单链和双链环介导的等温扩增(LAMP)分析检测鳗弧菌和溶藻弧菌。

通过针对VgroEL基因,从污染的海水中分离并同时开发了单双链和双链环介导的等温扩增(LAMP)分析方法,用于分别检测和同时检测受污染海水中的鱼类主要细菌病原体鳗弧菌和鱼类和人的病原体溶藻弧菌。鳗,其编码分子伴侣蛋白,并且fklB的基因溶藻弧菌,它编码一个22千道尔顿(kDa)的脯氨酰肽基异构酶。产生一致结果的最佳反应条件是groEL鳗弧菌为单)为65°C 30分钟,63°C 30分钟和63°C 40分钟,分别通过视觉检测方法(使用钙黄绿素和SYBR Green I)和琼脂糖凝胶电泳分析了fklB溶藻弧菌的)和groEL + flkB(双链)LAMP检测。发现该测定法是特定物种的,与弧菌属密切相关。未检测到。对于纯培养物和人工污染的海水中的DNA模板,LAMP测定的检测限(LoDs)为10和14 fg(groEL测定;对于鳗弧菌),12.5和17 fg(fklB测定;对于溶藻弧菌))和每个反应分别50和70 fg(双重测定),这比常规聚合酶链反应(PCR)的LoD要好得多。发现单重和双重LAMP测定法快速,物种特异性且对检测鳗弧菌溶藻弧菌敏感,适用于实验室和现场诊断。
更新日期:2019-05-16
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