Development of PCR techniques for the detection of Vibrio carchariaeand perkinsus olseni in abalone tissues

摘要

Global demand for abalone has significantly increased in recent years, however, wild stocks of abalone have declined in number, as a result of overexploitation and the spread of infectious disease. Disease outbreaks are recognised as a significant constraint to aquaculture production and trade that affects both economic development and the socio-economic revenue of many countries. Bacteria and protozoa are the most commonly encountered pathogens in abalone shellfish. Vibrio harveyi/carchariae is a highly pathogenic bacterium to abalone and Perkinsus atlanticus/olseni is a protozoan that also causes severe disease in this shellfish. Both pathogens are ranked amongst the top ten most significant disease causing organisms of abalone. In this study, a multiplex PCR method was developed to simultaneously amplify a 413 bp region of the 16S rRNA sequence of V. carchariae/harveyi and a 155 bp region of the actin mRNA gene sequence of Haliotis spp. This multiplex PCR was used to amplify these sequences in both fixed tissues and paraffin embedded tissues of infected Haliotis tuber-culata. A primer set was designed to target a 245 bp region of the ITS sequence of P. atlanticus from paraffin embedded samples of infected Ruditapes decussatus which could be adapted to detect P. olseni in abalone tissues. Also quantitative PCR using these primers is a further potential development. These PCR protocols offer a rapid and specific method for the identification of V. carchariae and P. olseni in shellfish.