Study On In Situ Hybridization Detection Method Of Clam Perkinsus Infection

Perkinsus infection can bring great damage to shellfishery and cause significant economic loses in the world, so it is an important quarantine loimia. Ray Fluid Thioglycolate Medium (RFTM) assay is a gold standard recommanded by the Office International Des Epizooties(OIE)to dectect the Perkinsus infection, but it can't differentiate Dinoflagellate with Perkinsus as the former has similar characters with later after cultured in Ray Fluid Thioglycolate Medium(RFTM). Besides, the PCR assay usually applied in entry-exit inspection of China has a high false positive rate. In this study, In situ hybridization(ISH) method to detect Perkinsus was established by using random primer to prepare Digoxigenin (DIG)-labeled Perkinsus probe for the first time. The detection of Digoxigenin (DIG)-labeled Perkinsus probe content rate was up to 100ng/μL. By southern blotting analysis, the probe was specific enough to distinguish Perkinsus from Bonamia and Haplosporidium and it was a universal probe which could be used in Perkinsus detection. Compared with RFTM assay, 20 samples of molluscs were detected by the developed ISH assay. Its diagonosis sensitivity and specificity were 90.9% and 100%, respectively.Shellfish samples collected from bays of the Huanghai Sea, including Ruditapes philippinarum, Meretrix lusoria, Cyclina sinensis and Concha ostreae, were detected by ISH. The result showed that Ruditapes philippinarum and Meretrix lusoria were infected by Perkinsus, while Cyclina sinensis and Concha ostreae were not infected. In conclusion, the in situ hybrization detection method of perkinsus, established in this study, could satisfy the need of entry-exit quarantine, and also be used to investigate Perkinsus infection in seashell cultivation farm.