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

Abstract

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°C, respectively. The amplification products were detected by electrophoresis. The detection limit of V. corallilyticus by LAMP was 3·6×103 CFU ml-1 (8 CFU per reaction), but PCR could detect up to 3·6×104 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. © 2010 The Society for Applied Microbiology.