DIFFERENTIAL DIAGNOSIS OF PERKINSUS SPECIES BY POLYMERASE CHAIN REACTION-RESTRICTION FRAGMENT LENGTH POLYMORPHISM ASSAY

A species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was performed to identify Perkinsus species. RFLP analysis of the rRNA ITS region using two restriction enzymes, RsaI HinfI, yielded species-specific fragment patterns that allowed successful identification of P. olseni, P. mediterraneus, P . marinus and P. chesapeaki. In order to validate the RFLP assay, the PCR products were cloned and sequenced. BLAST searches and phylogenetic analysis confirmed the specific identification carried out by RFLPs. Moreover, this method allows us to report for the first time the identification of P. olseni infecting the carpet-shell clams from the NW Adriatic Sea (Italy). The PCR-RFLP assay herein described may be useful to provide accurate, rapid and inexpensive identification of Perkinsus species. It may thus aid in ongoing epizootiological studies and diseases control programmes.