ORIGINAL ARTICLE Development of a haemolysin gene-based multiplex PCR for simultaneous detection of Vibrio campbellii, Vibrio harveyi and Vibrio parahaemolyticus S. Haldar 1 , S.B. Neogi 1 , K. Kogure 2 , S. Chatterjee 1 , N. Chowdhury 1 , A. Hinenoya 1 , M. Asakura 1,3 and S. Yamasaki 1 1 Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka, Japan 2 Ocean Research Institute, The University of Tokyo, Tokyo, Japan 3 Research and Development Centre, Fuso Pharmaceutical Industries Ltd., Osaka, Japan Introduction Vibrio spp. are naturally inhabiting aquatic bacteria and Vibrio campbellii is known to be associated with eco- nomically important shrimp farms. However, previously it was considered as nonpathogenic or might be mis- identified with the closest neighbour species Vibrio harveyi. Pathogenic potential among V. campbellii strains has been revealed in contemporary studies using crustacean model systems like Artemia franciscana, Litopenaeus vannamei etc (Sung et al. 2008). Vibrio harveyi and Vibrio parahaemolyticus are among the major Vibrio species responsible for diseases in many wild and reared aquatic organisms including shrimp, mollusks, fish and vertebrates (Thompson et al. 2004; Austin and Zhang 2006). Because of the plasticity of the Vibrio genomes with frequent homologous recombination in the marine envi- ronment, species boundaries are fragile (Fraser et al. 2007). Existence of high similarity among genes com- monly used for identification makes it problematic for differentiation of species belonging to the core clade of Vibrionaceae (e.g., V. campbellii, V. harveyi, V. parahae- molyticus). Sequencing of genes like 16S rRNA, 23S rRNA, 16S-23S intergenic spacer region or gyrB cannot differentiate these closely related species (Gomez-Gill Keywords haemolysin, multiplex PCR, Vibrio campbellii, Vibrio harveyi, Vibrio parahaemolyticus. Correspondence Shinji Yamasaki, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58, Rinkuorai-kita, Izumisano, Osaka 598-8531, Japan. E-mail: shinji@vet.osakafu-u.ac.jp Present address S. Haldar, Marine Biotechnology and Ecology Discipline, Central Salt and Marine Chemicals Research Institute (CSIR), G.B. Marg, Bhavnagar-364021, Gujarat, India. 2009 ⁄ 1673: received 20 September 2009, revised 21 October 2009 and accepted 24 October 2009 doi:10.1111/j.1472-765X.2009.02769.x Abstract Aim: To develop a haemolysin (hly) gene-based species-specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahae- molyticus. Methods and Results: The complete hly genes of three V. campbellii strains iso- lated from diseased shrimps were sequenced and species-specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species. Significance and Impact of the Study: Because there is lack of simple, rapid and cost-effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view. Letters in Applied Microbiology ISSN 0266-8254 146 Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 50 (2010) 146–152 ª 2009 The Authors