USE OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION ASSAY FOR THE DETECTION OF VIBRIO ANGUILLARUM O2β, THE CAUSATIVE AGENT OF VIBRIOSIS IN ATLANTIC COD, GADUS MORHUA

A loop-mediated isothermal amplification (LAMP)-based assay for the detection of Vibrio anguillarum O2 beta, the causative agent of vibriosis in Atlantic cod, Gadus morhua, was developed. Five sets of primers targeting the flanking regions of the genes, hemolysin and amiB, which encodes the peptidoglycan hydrolase N-acetylmuramoyl-L-alanine amidase of the pathogen were designed. The primers were specific for the detection of Vibrio anguillarum O2 beta with no cross reactions to other bacterial pathogens commonly infecting Atlantic cod, e.g., Yersinia ruckeri, Francisella piscicida, Aeromonas salmonicida and some endogenous bacteria found in the gut of Atlantic cod. The detection limit of the assay was 10 pg of bacterial DNA/mL or 10 fg of bacterial DNA per LAMP reaction; however, the sensitivity of the reaction decreased by 3-log dilution in the presence of 1 mg/mL of mucus samples as inhibitors. Nevertheless, the assay can be potentially used as a direct and nondestructive method to detect the pathogen in the fish, thus making the assay less time-consuming. These results suggest that the LAMP assay is a specific and sensitive molecular method to detect vibriosis in Atlantic cod and will be helpful for routine surveillance programs in aquaculture systems. PRACTICAL APPLICATIONS Loop-mediated isothermal amplification (LAMP), a potential diagnostic technique performed under isothermal conditions without the use of any sophisticated equipment, can be used for the rapid and early detection of Vibrio anguillarum 02 beta causing vibriosis in Atlantic cod. The developed assay is highly sensitive and can be used to detect the pathogen prior to the onset of infection, thus, immediate measures can be applied to prevent heavy losses of the cultured stock.