Development of a PCR-ELISA assay for diagnosis of Perkinsus marinus and Perkinsus atlanticus infections in bivalve molluscs

Abstract

Perkinsus atlanticus and P. marinus have been associated with mass mortality of bivalve molluscs. Perkinsus infections are routinely diagnosed by histology or the fluid thioglycollate medium (FTM) assay. In this study, we describe the development of a PCR-enzyme-linked immunosorbent assay (ELISA) for amplification and rapid detection of Perkinsus species. The PCR reactions were selected to either amplify an IGS sequence region shared by currently accepted Perkinsus species or to simultaneously amplify IGS regions specific to either P. atlanticus or P. marinus. The specific hybridisation of DIG-labelled amplified products to species-specific capture probes was detected colorimetrically. This assay is able to specifically detect P. atlanticus and P. marinus, and the intensity of the colorimetric signal is dependent upon the amount of amplified product. The PCR-ELISA assay format is 100-fold more sensitive than visualisation of PCR products on ethidium bromide (EtdBr)-stained agarose gels, and as sensitive as Southern hybridisation. The sensitivity limit of PCR-ELISA was 1 pg of DNA from P. atlanticus. No cross-reactivity of the assay was observed against the host DNA. When applied to the detection of P. atlanticus in clams, 39 samples out of 45 yielded concordant results for FTM assay and PCR-ELISA detection.