Development of a quadruplex loop-mediated isothermal amplification assay for field detection of four Vibrio species associated with fish disease.

A quadruplex loop-mediated isothermal amplification (LAMP) method was developed to detect four Vibrio species, including Vibrio ichthyoenteri, Vibrio parahaemolyticus, Vibrio scophthalmi, and Vibrio vulnificus, simultaneously. Four sets of species-specific primers were designed with different restriction sites contained in the inner primers. The quadruplex LAMP method could distinguish four Vibrio species via the subsequent restriction enzyme analysis. The sensitivity of the quadruplex LAMP method were 10(2)-10(3) times higher than the sensitivity of conventional PCR. V. scophthalmi, V. vulnificus, V. parahaemolyticus and V. ichthyoenteri could be detected in the different tissues of the infected fish by the quadruplex LAMP method simply and conveniently through using SYBR Green I to facilitate visual inspection of the LAMP products. The method we developed in this study could be a simple and convenient diagnostic tool for field detection of Vibrio infection in fish.