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A quadruplex loop-mediated isothermal amplification (LAMP) method was developed to detect four  Vibrio  species, including  Vibrio ichthyoenteri ,  Vibrio  parahaemolyticu s,  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. parahaemolyticu s 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.

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