Multiplex PCR using YeaD and 16S rRNA gene to identify major pathogens in vibriosis of Litopenaeus vannamei

The Vibrio species causing major diseases in Litopenaeus vannamei are Vibrio harveyi, Vibrio alginolyticus, and Vibrio parahaemolyticus. For multiplex PCR primers, YeaD was used to detect the three Vibrio species. Bioinformatic analysis such as MultiPLX and primer-BLAST was used to design stable and species-specific multiplex PCR primers. Multiplex PCR results showed clear band patterns with bands at 185bp for V. alginolyticus, 396bp for V. harveyi, 805bp for V. arahaemolyticus, and 596bp for common Vibrio species. The minimum concentration of DNA was measured by PCR; the value for V. alginolyticus was 0.1ng, that of V. harveyi was 0.03ng, and that of V. parahaemolyticus was 0.003ng. Taken together, YeaD showed stability and specificity in identifying Vibrio species. Our multiplex PCR amplification method is an effective and inexpensive tool for identifying Vibrio species.