Detection of Vibrio anguillarum and Vibrio alginolyticus by Singleplex and Duplex Loop-Mediated Isothermal Amplification (LAMP) Assays Targeted to groEL and fklB Genes
| Autor: | In-Soo Kong, Jong Min Lee, Mahbubul Pratik Siddique, Tawheed Hasan, Won Je Jang, Chang-Hoon Kim |
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| Rok vydání: | 2019 |
| Předmět: |
Microbiology (medical)
Vibrio anguillarum Loop-mediated isothermal amplification Microbiology law.invention Fish Diseases 03 medical and health sciences chemistry.chemical_compound Bacterial Proteins law Animals Humans Vibrio alginolyticus Polymerase chain reaction Vibrio 030304 developmental biology 0303 health sciences biology 030306 microbiology Chemistry Chaperonin 60 biology.organism_classification Molecular biology GroEL Vibrio Infections Agarose gel electrophoresis SYBR Green I Nucleic Acid Amplification Techniques |
| Zdroj: | International Microbiology. 22:501-509 |
| ISSN: | 1618-1905 1139-6709 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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