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Diseases of Aquatic Organisms

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DAO 151:111-121 (2022)  -  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. Piesz1, Abigail K. Scro2, Ryan Corbett1, Kathryn Markey Lundgren2,3, Roxanna Smolowitz2, Marta Gomez-Chiarri1,*

1Department of Fisheries, Animal, and Veterinary Science, University of Rhode Island, Kingston, Rhode Island 02881, USA
2Aquatic Diagnostic Laboratory, Roger Williams University, Bristol, Rhode Island 02809, USA
3National Cold Water Marine Aquaculture Center, USDA, CBLS 469, Kingston, Rhode Island 02881, USA
*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 H. 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 on 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 and 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 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.


KEY WORDS: Crassostrea virginica · Eastern oyster · Diagnostic assay · Perkinsus marinus · Haplosporidium nelsoni · Haplosporidium costale · qPCR


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Cite this article as: Piesz JL, Scro AK, Corbett R, Markey Lundgren K, Smolowitz R, Gomez-Chiarri M (2022) Development of a multiplex qPCR for the quantification of three protozoan parasites of the eastern oyster Crassostrea virginica. Dis Aquat Org 151:111-121. https://doi.org/10.3354/dao03694

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