Journal
JOURNAL OF FISH DISEASES
Volume 44, Issue 12, Pages 1993-2001Publisher
WILEY
DOI: 10.1111/jfd.13513
Keywords
Aeromonas salmonicida; hydroxynaphthol blue dye; loop-mediated isothermal amplification; vapA gene; visual detection
Funding
- Major Agricultural Applied Technological Innovation Projects of Shandong Province, Fish Innovation Team of Shandong Agriculture Research System [SDAIT-12-06]
- Aquatic Animal Immunologic Agents Engineering Research Center of Shandong Province
- National Natural Science Foundation of China [32002421]
- Advanced Talents Foundation of QAU [6651118016]
- Natural Science Foundation of Shandong Province [ZR2019BC009]
- Key Research and Development Program in Shandong [2018YFJH0703]
- Breeding Plan of Shandong Provincial Qingchuang Research Team (2019)
- First class fishery discipline' programme in Shandong Province, special top talent plan ` One Thing One Decision (Yishi Yiyi)'
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The study developed a rapid detection method, LAMP-HNB assay, for Aeromonas salmonicida, an important pathogen in fish farming, with good specificity and high sensitivity. The method has potential applications in diagnostic testing, health certification, and surveillance programs.
To make crucial prevention, reduce fish losses and minimize the economic damage of diseases on the fish farm owners, a rapid detection of fish pathogens is mandatory. In this study, a loop-mediated isothermal amplification assay combined with hydroxynaphthol blue dye (LAMP-HNB) was developed and used for the rapid detection of Aeromonas salmonicida that caused significant economic losses in fish farming. Firstly, a pair of outer and inner primers specific for conserved fragment of vapA gene in A. salmonicida were designed and synthesized. Secondly, by optimizing the reaction conditions including reaction temperature, time, Mg2+ concentration, dNTP concentration and primer ratio, a LAMP-HNB assay was successfully established for the detection of A. salmoncida. Thirdly, the assay showed good specificity with no false-positive and false-negative results, and good sensitivity with the detection limit of 3.077 x 10(-6) ng/mu l, which was 10(2) times more sensitive than the conventional PCR. Finally, the LAMP-HNB assay was validated by the fish samples inoculated with different concentrations of A. salmoncida. This is the first development of rapid visual detection of A. salmonicida based on LAMP-HNB assay, which has great application prospect and market for diagnostic testing, health certification and active surveillance programmers.
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