Characterization of the ribosomal RNA locus of Perkinsus atlanticus and development of a polymerase chain reaction-based diagnostic assay

The rRNA locus of Perkinsus atlanticus from the clam Ruditapes decussatus c ultivated on the Atlantic coast of Spain was cloned and sequenced. Sequence s of the internal transcribed spacer (ITS) from the rRNA locus were compare d to sequences reported earlier for a P. atlanticus isolate from Portugal a nd to those from other Perkinsus species. The ITS 1 sequence of the Spanish P. atlanticus isolate was identical to the Portuguese P. atlanticus sequen ce and had 76.6% identity to the ITS1 of Perkinsus marinus. The ITS2 sequen ce had 99.7% identity to the Portuguese P. atlanticus ITS2, 92.5% identity to the P. marinus ITS2, and 99.5% identity to the Perkinsus olseni ITS2. We report for first the time the small subunit (SSU) and nontranscribed space r (NTS) of P. atlanticus. The P. atlanticus SSU sequence was 99.6% identica l to that of an unidentified Perkinsus species from the Australian clam Ana dara trapezia and 98.0% identical to that of P. marinus. Further, our resul ts support the proposal that P. atlanticus, P. olseni, and the Perkinsus sp . from A. trapezia constitute a subgroup of Perkinsus species distributed i n the Pacific and eastern Atlantic, different from P. marinus that is distr ibuted along the western edge of the Atlantic. Based on the NTS sequence of P. atlanticus from Spain and the differences with P. marinus NTS (62.2% id entity), we developed a polymerase chain reaction (PCR)-based diagnostic as say with a lowest limit of detection of 0.01 amol of cloned NTS DNA as asse ssed on ethidium bromide-stained agarose gels. Specificity of the PCR-based assay was tested with samples from the clams R. decussatus, Ruditapes phil ippinarum, and Venerupis pullastra collected in P. atlanticus-enzootic area s of Spain. The specificity and sensitivity demonstrated for this NTS-based PCR assay validate its use as a tool for assessment of P. atlanticus in mo lluscs.