A quantitative competitive polymerase chain reaction assay for the oyster pathogen Perkinsus marinus

A quantitative competitive polymerase chain reaction (QCPCR) assay was deve loped for the oyster parasite Perkinsus marinus. PCR primers for the rRNA g ene region of P. marinus amplified DNA isolated from P. marinus but not fro m Perkinsus atlanticus, Crassostrea virginica, or the dinoflagellates Perid inium sp., Gymnodinium sp., or Amphidinium sp. A mutagenic primer was used to create a competitor plasmid molecule identical to the P. marinus target DNA sequence except for a 13-bp deletion. Both P. marinus and competitor DN A amplified with equivalent efficiencies. Each of 25 oysters was processed by 5 P. marinus diagnostic methods-Ray's fluid thioglycollate medium (FTM) tissue assay, FTM hemolymph assay, whole oyster body burden assay, QCPCR of combined gill and mantle (gill/mantle) tissue, and QCPCR of hemolymph. The QCPCR assay enabled detection of 0.01 fg of P. marinus DNA in 1.0 mu g of oyster tissue. QCPCR of gill/mantle tissue or hemolymph as well as the body burden assay detected infections in 24 of 25 oysters. Ray's FTM tissue ass ay detected only 19 infections. The FTM hemolymph assay detected only 22 in fections. Regression analysis of QCPCR results and FTM results indicated th at the QCPCR assays were effective in quantitating P. marinus infections in oyster tissues.