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Rapid, sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification

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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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Authors and Affiliations

  1. Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071, China

    Hongwei Gao  (高宏伟), Fuhua Li  (李富花), Xiaojun Zhang  (张晓军), Bing Wang  (王兵) & Jianhai Xiang  (相建海)

  2. Shandong Entry-Exit Inspection and Quarantine Bureau of the People’s Republic of China, Qingdao, 266001, China

    Hongwei Gao  (高宏伟)

Authors
  1. Hongwei Gao  (高宏伟)
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  2. Fuhua Li  (李富花)
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  3. Xiaojun Zhang  (张晓军)
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  4. Bing Wang  (王兵)
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  5. Jianhai Xiang  (相建海)
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Corresponding author

Correspondence to Jianhai Xiang  (相建海).

Additional information

Supported by the National Basic Research Program of China (973 Program) (No. 2006CB101804), the General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (No. 2007IK167), and National Department Public Benefit Research Foundation (No. 200803012)

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Gao, H., Li, F., Zhang, X. et al. Rapid, sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification. Chin. J. Ocean. Limnol. 28, 62–66 (2010). https://doi.org/10.1007/s00343-010-9247-3

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  • DOI: https://doi.org/10.1007/s00343-010-9247-3

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