Title: Development of a two-step, non-probed multiplex real-time PCR for surveilling Vibrio anguillarum in seawater Authors
|Hickey, Michael -|
|Lee, Jung-Lim -|
Submitted to: Journal of Fish Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 23, 2014
Publication Date: June 1, 2015
Citation: Hickey, M.E., Richards, G.P., Lee, J. 2014. Development of a two-step, non-probed multiplex real-time PCR for surveilling Vibrio anguillarum in seawater. Journal of Fish Diseases. 38, 551-559. Interpretive Summary: A marine bacterium known as Vibrio anguillarum causes illness and death in a variety of fish in both aquaculture and natural settings. An improved method was developed to detect V. anguillarum in seawater. The method is more rapid, sensitive and specific than other methods and relies on the detection of DNA corresponding to specific genes found in V. anguillarum. This test can detect as few as five V. anguillarum per ml of seawater in 80 min compared to cultural methods which take 3 days. Rapid detection of V. anguillarum in aquaculture facilities is critical for effective treatment of infected fish in order to reduce mortalities.
Technical Abstract: Vibrio anguillarum is an aggressive and halophilic bacterial pathogen commonly found in seawater. Its presence in aquaculture facilities causes significant morbidity and mortality among aquaculture species primarily from hemorrhaging of the body and skin of the infected fish that eventually leads to death, collectively recognized as the disease vibriosis. In the present study, a non-probe, multiplex SYBR Green quantitative PCR (MSG qPCR) assay to rapidly detect V. anguillarum presence in seawater was developed. Specific primers targeting genes vah1, empA, and rpoN of V. anguillarum were selected for multiplex-reaction among 11 different primer sets and the extension step was eliminated. Primer concentration, denaturation time, as well as annealing time and temperature of DNA amplification were optimized significantly, thus reducing reaction-duration. Culture-based methods for V. anguillarum detection have taken as long as three days while MSG qPCR, as described in this study, detects as few as 5 CFU of V. anguillarum per ml of seawater in 80 min with molecular precision and with melting curve confirmation. This method will allow for the timely detection of V. anguillarum in aquaculture systems, faster treatment of infected fish, and reduced losses to the aquaculture industry.