Establishment And Application Of The Detection Methods Of Shellfish Perkinsosis

Perkinsus infection of marine bivalves and oysters can have severe pathogenic effect on their hosts and cause significant economic loses to the aquatic farming industry in the world. It was reported that Perkinsus infection of mollusks has a broad geographic range including China.In this study, the Manila clam Ruditapes philippinarum was choose as a research target, which was widely cultured at the coast of China. In the research basis of the development of the ray's fluid thioglycollate medium (RFTM) culture method of Perkinsus, the real time TaqMan PCR detection method was developed considering its high speed, sensitivity and specificity. The primers and TaqMan probe were designed and chose to amplify the conserved internal transcripted spacer 2 (ITS-2) segment of genus Perkinsus ribosomal DNA. Results indicated that the dynamic range of the developed method was from 1.0×101 copies to 1.0×107 copies plasmid DNA, and the detection limit was 100 copies of plasmid DNA artificially contaminated to clam tisue. The developed method was specific enough to distinguish Perkinsus from Bonamia spp and other protistan parasites while not interfered by mollusk tissue DNA. Selecting RFTM culture as gold standard, the diagnostic sensitivity and specificity of the developed method were 93.81% and 84.21% respectively. 16 samples including Ruditapes philippinarum, Meretrix lusoria,Sinonovacula constricat canarck,collected from the Huanghai bays of liaoning Province, were subjected to RFTM culture and real-time PCR detetcion. As result, the Perkinsus infection status was determined in Ruditapes philippinarum and Meretrix lusoria, while not in Sinonovacula constricat canarck. Perkinsus isolated in this study was classified as P. olseni by Phylogenetic tree analysis of the related ITS2 DNA sequences and further verified by the deveoped P. olseni specific real time TaqMan PCR assay.In conclusion, the real time TaqMan PCR detection method of Perkinsus was developed in China. Using RFTM culture combining with the developed real time TaqMan PCR, the Perkinsus infection status could be conformed. Perkinsus isolates were classified as P. olseni by Phylogenetic tree analysis of the related ITS2 DNA sequences and further verified by the deveoped P. olseni specific real time TaqMan PCR. The developed method can be used to investigate Perkinsus in the farm and for quarantine use.