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

Shun Zhou; Zhi-Xin Gao; Min Zhang; Dan-Yang Liu; Xin-Peng Zhao; Yong Liu

doi:10.1186/s40064-016-2760-x

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

Springerplus. 2016 Jul 16;5(1):1104. doi: 10.1186/s40064-016-2760-x. eCollection 2016.

Authors

Shun Zhou^#^{1

2}, Zhi-Xin Gao^#¹, Min Zhang^#¹, Dan-Yang Liu¹, Xin-Peng Zhao¹, Yong Liu¹

Affiliations

¹ Marine Science and Engineering College, Qingdao Agricultural University, Qingdao, 266109 People's Republic of China.
² College of Fisheries, Qingdao Ocean University, Qingdao, 266100 People's Republic of China.

^# Contributed equally.

Abstract

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.

Keywords: A quadruplex LAMP method; Field detection; Vibrio ichthyoenteri; Vibrio parahaemolyticus; Vibrio scophthalmi; Vibrio vulnificus.