Digital PCR (dPCR) vs. Quantitative PCR (qPCR) approaches for quantification of two Perkinsus species within clam tissue samples.

Abstract

The parasite Perkinsus olseni (Perkinsea, Alveolata), the etiological agent of Perkinsosis, infects a wide range of bivalves and gastropods, including clams, particularly in Europe. This parasite coexists in sympatry with another Perkinsus species, P. chesapeaki, which, as opposed to P. olseni, has not been directly associated to mortality events. Accurate detection and quantification of Perkinsus infections, even at low infection intensities, are crucial for monitoring clam population health and assessing risks associated with emerging diseases. In this study, we compared molecular methodologies based on duplex real-time quantitative PCR (qPCR) and digital PCR (dPCR) in order to develop effective host resource management strategies. We first evaluated detection capabilities and the impact of potential inhibitors using both methodologies across varying DNA concentrations. Subsequently, we applied these methods to two contrasting French environments: Noirmoutier, characterized by low prevalence and infection intensity, and Arcachon Bay, which exhibits high prevalence and infection intensity. Our results demonstrate that dPCR should be prioritized for detecting and quantifying parasites at low infection intensities (10¹-10² cp.µL^-1), as it might minimize false-negative results compared to qPCR. Notably, dPCR provided new insights and revealed cryptic infections, demonstrating greater efficiency in detecting P. chesapeaki in lightly infected sites such as Noirmoutier. Conversely, infection intensity was underestimated with dPCR relative to qPCR for clams with moderate to high Perkinsus infection levels (10³ cp.µL^-1 or higher), proving the latter more suitable for medium to heavily infected areas like Arcachon Bay. These findings are important as they represent the first in situ monitoring of both Perkinsus species using culture-free methodologies. This work provides essential tools for resource management and conservation strategies to address emerging diseases.