Loop-mediated isothermal amplification method for rapid detection of Vibrio alginolyticus, the causative agent of vibriosis in mariculture fish.

Abstract

AIMS: The purpose of this study was to develop a loop-mediated isothermal amplification (LAMP) method for the rapid, sensitive and simple detection of Vibrio alginolyticus in mariculture fish. METHODS AND RESULTS: LAMP primers were designed by targeting the gyrB gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64 degrees C in a simple water bath. The detection sensitivity of the LAMP assay for the detection of V. alginolyticus is about 3.7 x 10(2) CFU ml(-1) (3.7 CFU per reaction). LAMP products could be judged with agar gel or naked eye after the addition of SYBR Green I. There were no cross-reactions with other bacterial strains indicating a high specificity of the LAMP. The LAMP method was applied to detect V. alginolyticus-infected fish tissues effectively. CONCLUSIONS: The LAMP established in this study is a simple, sensitive, specific, inexpensive and rapid protocol for the detection of V. alginolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: This LAMP method provides an important diagnostic tool for the detection of V. alginolyticus infection both in the laboratory and field.