Molecular methods for the diagnosis of Marteilia refringens

A nested polymerase chain reaction (PCR) has been developed for the diagnos is of Marteilia refringens in mussels (Mytilus galloprovincialis) from Gali cia (NW Spain). Specific primers were used in two consecutive amplification reactions, that were able to detect 15 fg of Marteilia DNA. The nested PCR yielded a 550 bp fragment that was used to generate a non-radioactive prob e, specific for Marteilia, as determined by in situ hybridization. This pro be was also used in a dot-blot developed for the diagnosis of the parasite. This method alone was much less sensitive than the nested PCR, however, it s use after the nested PCR significantly increased the sensitivity of the P CR. When comparing these molecular diagnosis tools with traditional diagnos is methods (smears and histology), we determined that the nested PCR could detect Marteilia in 65% of the mussels screened, while only 20-25% of the m ussels were identified as infected by traditional methods.