A PCR-ELISA method for direct detection of the oyster pathogen Haplosporidium nelsoni

A rapid method, utilizing both polymerase chain reaction (PCR) and enzyme-l inked immunosorbent assay (ELISA), was developed for detection of oyster MS X disease. The technique included using Haplosporidium nelsoni pathogen-spe cific PCR primers (based on ribosomal RNA genes), a Chelex resin (for rapid DNA extraction from oyster mantle tissues), and cloned H. nelsoni rRNA pla smid DNA (for use as a capture probe). Digoxigenin was incorporated into th e pathogen-specific PCR products, which were captured by the coated probe i n a fast hybridization reaction and then detected by ELISA. The sensitivity of PCR amplification on cloned plasmid DNA was 10 fg for detection by stai ned agarose gel, and increased to 0.01 fg for ELISA. Positive signals were observed in infected oysters using the PCR-ELISA technique. This method may be applicable to early detection of infection.