Real-time PCR for Vibrio anguillarum Detection in Maintaining Seawater Quality of Aquaculture

Introduction: Vibrio anguillarum is a well-known opportunistic bacterial pathogen that causes vibriosis in a range of marine organisms around the world. Vibriosis infection of aquaculture or wild fish has been reported as a detrimental factor of fishery yields and financial stability to the industry. As such, measures of V. anguillarum-control via vaccination, phage therapy, and chemical sterilization of aquaculture waters are exhibited to maintain healthy seafood yields and commercial sales. Alongside growing consumer awareness of the ethics involved in mass food production and related practices are an increased demand for natural, organic methods of agricultural commerce.

Purpose: In order to contribute to this growing industrial trend, the present study served to develop a rapid method of pathogenic detection that will allow technicians the opportunity to better evaluate the quality of aquaculture waters based on virulent V. anguillarum levels therein.

Methods: Four conserved virulence genes in V. anguillarum were screened for amplification efficiency via PCR in real-time and conditions such as cycle repetition, reagent concentration, reaction duration, and temperature of amplification were optimized.

Results: Conventional methods of pathogenic detection can take several hours, require additional enrichment strategies for low bacterial levels, and do not produce quantifiable data. By simultaneously targeting three known virulence genes in the V. anguillarum genome, the real-time PCR set forth in this study allows for the quantification of pathogenic-specific V. anguillarum populations in less than 70 minutes and is able to detect as little as 3 CFU ml^-1 V. anguillarum in seawater; without the need for additional enrichment or electrophoresis steps for data analysis.

Significance: This protocol can be used to enhance standard food safety operations in the aquaculture industry by offering technicians quantifiable proof that some aquaculture tanks can be exempt from vaccination procedures that will promote natural cultivation of fish for consumer safety and satisfaction.