Application of the specific gene for the detection of Vibrio anguillarum by Polymerase Chain Reaction

Abstract

Vibrio anguillarum, an opportunistic fish pathogen, is the main species responsible for vibriosis, a disease that affects feral fish, farmed fish, and causes considerable economic losses in marine aquaculture. Several studies aimed at detecting V. anguillarum have targeted ribosomal DNA belong to the bacterium. In this study, we used polymerase chain reaction (PCR) and targeted the amiB gene, encodes the peptidoglycan hydrolase N-acetylmuramoyl-L-alanine amidase, and rpoS gene, a general stress regulator, to detect V. anguillarum. PCR specificity was identified by 429-bp and 689-bp amplification of 6 strains of V. anguillarum and by the loss of a PCR product with 36 other bacterial species. The PCR produced a 429-bp amplified fragment from as little as 1 pg of V. anguillarum DNA. The limit of detection for this PCR technique was approximately 20 bacterial colonies in 25 mg of infected flounder tissue. And the PCR assay was sensitive enough to detect rpoS expression from 3 pg of genomic DNA, or from 6 colony-forming units mL-1 (cfu) of cultured bacterium. However, the assay was less sensitive when genomic DNA from infected founder and prawn was used (limit of detection, 50 ng and 10 ng per gram tissue, respectively). These data demonstrated that PCR amplification of amiB and rpoS gene is a sensitive, species-specific and rapid method to detect V. anguillarum in practical applications.