Duplex droplet digital PCR enables simultaneous quantification of algal giant virus DSLLAV1 and virophage DSLV8 in natural and laboratory samples.

Abstract

Cell-virus-virophage (CVv) systems involve virophages parasitizing giant viruses within eukaryotic hosts, forming unique virus-virus interactions with complex ecological implications. However, quantitative tools for studying such systems-particularly in freshwater algae-remain limited. In this study, we developed and optimized a duplex droplet digital PCR (ddPCR) assay to simultaneously detect and quantify Dishui Lake Large Algal Virus 1 (DSLLAV1), a Mimiviridae-like algal giant virus, and its associated Dishui Lake virophage 8 (DSLV8) in the Dishui Lake ecosystem. Target-specific primers and TaqMan probes were designed based on viral genomic sequences, and assay conditions were optimized for annealing temperature, primer/probe concentrations, and droplet separation. The established assay demonstrated high specificity and sensitivity, with detection limits of 0.13 and 0.16 copies/µL for DSLLAV1 and DSLV8, respectively. The method outperformed qPCR in sensitivity and maintained stability across environmental and infection-derived samples. This ddPCR method provides a robust platform for monitoring virus-virophage dynamics and offers new opportunities for investigating the ecological and evolutionary roles of CVv systems in aquatic environments.