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

Abstract

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. 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. The LAMP established in this study is a simple, sensitive, specific, inexpensive and rapid protocol for the detection of V. alginolyticus. This LAMP method provides an important diagnostic tool for the detection of V. alginolyticus infection both in the laboratory and field.