Rapid, sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification |
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| Authors: | Hongwei Gao Fuhua Li Xiaojun Zhang Bing Wang Jianhai Xiang |
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| Institution: | (1) Department of Cell and Molecular Biology, University of Rhode Island, 02881 Kingston, RI, USA;(2) Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, 615 N. Wolfe Street, 21205 Baltimore, MD, USA;(3) Department of Medicine,LRB 360D, University of Massachusetts Medical School, 364 Plantation Street, 01605 Worcester, MA, USA |
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| Abstract: | Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection
and identification of V. anguillarum, and other pathogens that infect marine organisms, is crucial to effective disease management. In this study, we developed
a loop-mediated amplification (LAMP) assay to detect V. anguillarum in an hour in a single tube without the need for thermal cycling. Conserved regions of the metalloproteinase (empA) gene of V. anguillarum served as the targets for primer design. A fragment of the empA gene was amplified at 65°C in the presence of the primer mixture and Bst DNA polymerase. In the optimized LAMP assay, 6.7 pg of V. anguillarum DNA could be detected. Six strains of V. anguillarum and 17 strains of non-V. anguillarum bacteria were used in this study to evaluate the species specificity of the primers. The six V. anguillarum strains gave a positive result in the LAMP assay. This method was also validated in V. anguillarum-infected fish. This LAMP method is more sensitive than PCR in the detection of V. anguillarum and shows good species specificity. The LAMP assay is therefore an effective method for the quick detection of V. anguillarum both in the laboratory and in the field. |
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