Abstract:Perkinsus sp. are parasites for marine bivalves. They can have severe pathogenic effect on their hosts and cause significant economic loses to the bivalve culture industry. To facilitate rapid and sensitive diagnosis of Perkinsus sp. the real time TaqMan PCR method was developed to detect Perkinsus sp. infection in oysters and other bivalves. The primers and TaqMan probe were designed and chosen to amplify the conserved internal transcript spacer 2 (ITS-2) segment of genus Perkinsus sp. ribosomal DNA. The sensitivity and specificity of the developed method were examined using the plasmid containing the targeted ITS-2 fragment as template. The online BLAST analysis showed that the primers and probe were specific enough to distinguish Perkinsus sp. from Bonamia sp. and other protistan parasites while not affected by bivalve tissue DNA at the same time. The dynamic range of the developed method was between 2.6×101and 2.6×107copies, meaning that the target DNA could be reliably detected and quantified for plasmid template DNA at a concentration as low as 26 copies. Thirty samples, collected from different sea areas of China, were tested for Perkinsus infection and 3 samples, collected from Shandong and Fujian coastal areas respectively, gave positive results. This test result was confirmed by traditional FTM culturing method. In conclusion, the real time PCR method is rapid, sensitive and specific for Perkinsus, and can be used to inspect Perkinsosis in the farm and for quarantine use.
