Sensitive and rapid detection of Vibrio corallilyticus by loop-mediated isothermal amplification targeted to the alpha subunit gene of RNA polymerase

Aims: A diagnostic protocol was developed for rapid detection of Vibrio corallilyticus by method of loop-mediated isothermal amplification (LAMP). Methods and Results: For cloning and sequencing of rpo A gene of V. corallilyticus, a set of four LAMP primers were designed by targeting the rpoA gene. With Bst DNA polymerase, the reaction time and temperature were optimized for 70 min at 65 degrees C, respectively. The amplification products were detected by electrophoresis. The detection limit of V. corallilyticus by LAMP was 3 center dot 6x103 CFU ml-1 (8 CFU per reaction), but PCR could detect up to 3 center dot 6x104 CFU ml-1 (72 CFU per reaction). The LAMP method was ninefold more sensitive than conventional PCR. The results also indicated that the LAMP reaction was highly specific to V. corallilyticus. Conclusions: The LAMP assay was a sensitive, specific and cost-effective method for the rapid detection of V. corallilyticus. Significance and Impact of the Study: This LAMP method provides an important diagnostic tool for the detection of V. corallilyticus infection. It can replace laborious biochemical tests for the identification of V. corallilyticus.