Differential diagnosis of Perkinsus species by polymerase chain reaction-restriction fragment length polymorphism assay

Perkinsosis is an infection of marine molluscs caused by the protistan parasites of the genus Perkinsus, which has been classified by the OIE as a disease that warrants notification. In the present study, we have applied a molecular genetic approach to develop an optional method for the specific identification of Perkinsus species.A species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay of the rRNA ITS region was developed to identify and distinguish among Perkinsus species. A taxonomic key was established that allows successful identification of Perkinsus species using a single restriction enzyme (Rsa I) to discriminate P. chesapeaki and P. marinus or by a combination of two endonucleases (Rsa I plus Hinf I) to discriminate P. olseni and P. mediterraneus. In order to validate the RFLP assay, the PCR products were cloned and sequenced, and its phylogenetic affinity was determined. Phylogenetic analysis confirmed the specific identification carried out by RFLPs. Herein is the first report of P. olseni in Manila clams from the NW Adriatic Sea (Italy), which we identified by employing this method. The PCR-RFLP assay herein described may be useful to provide accurate, rapid and inexpensive identification of Perkinsus species, and may aid in ongoing epizooetiological studies and diseases control programmes.