novel multiplex PCR method for detecting virulent strains of Vibrio alginolyticus

The bacterial strains obtained from various origins were tested with the novel primers targeting the collagenase gene, ompK gene and toxR gene to establish a multiplex polymerase chain reaction (PCR) method. These primers successfully recognized all virulent strains of Vibrio alginolyticus, but the avirulent strains were not recognized by the multiplex PCR because of lack of the collagenase and toxR genes. In a 50 μL multiplex PCR mixture, the lowest detection limit is 8.8 x 10² cells of virulent strains of V. alginolyticus. The multiplex PCR method was successfully developed to identify virulent strains of V. alginolyticus, and provides a rapid, sensitive, specific and reliable technology for diagnosing virulent strains of V. alginolyticus. Therefore, the novel multiplex PCR in the present paper can be useful for any laboratory working with vibriosis detection of aquatic animals.