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Diseases of Aquatic Organisms

    DAO prepress abstract   -  DOI: https://doi.org/10.3354/dao03694

    Development of a multiplex qPCR for the quantification of three protozoan parasites of the eastern oyster Crassostrea virginica

    Jessica L. Piesz, Abigail K. Scro, Ryan Corbett, Kathryn Markey Lundgren, Roxanna Smolowitz, Marta Gomez-Chiarri*

    *Corresponding author:

    ABSTRACT: A multiplex quantitative PCR (qPCR) assay for the simultaneous detection of 3 eastern oyster Crassostrea virginica parasites, Perkinsus marinus, Haplosporidium nelsoni, and Haplosporidium costale, was developed using 3 different fluorescently labeled hydrolysis probes. The primers and probe from a previously validated singleplex qPCR for P. marinus detection were combined with newly designed primers and probes specific for H. nelsoni and H. costale. The functionality of the multiplex assay was demonstrated in 2 different platforms by the linear relationship of the standard curves and similar cycle threshold (CT) values between parasites. Efficiency of the multiplex qPCR assay on the Roche and BioRad platforms ranged between 93-101%. The sensitivity of detection ranged between 10 and 100 copies of plasmid DNA for P. marinus and Haplosporidium spp., respectively. The concordance between the Roche and BioRad platforms in the identification of the parasites P. marinus, H. nelsoni, and H. costale was 91%, 97%, and 97%, respectively, with a 10-fold increase in the sensitivity of detection of Haplosporidium spp. on the BioRad thermocycler. The concordance between multiplex qPCR and histology for P. marinus, H. nelsoni, and H. costale was 54, 57, and 87%, respectively. Discordances between detection methods were largely related to localized or low levels of infections in oyster tissues, and qPCR was shown to be the more sensitive diagnostic. The multiplex qPCR developed here is a sensitive diagnostic tool for the quantification and surveillance of single and mixed infections in the eastern oyster.