A novel digital PCR assay for detection and comprehensive characterization of Molluscum contagiosum virus genotypes MOCV1, MOCV2, and MOCV3 and recombinant lineages.

Molluscum contagiosum virus (MOCV) is an important human pathogen causing a high disease burden worldwide. It is the last exclusively human-infecting poxvirus still circulating in its natural reservoir-a valuable model of poxviral evolution. Unfortunately, MOCV remains neglected, and little is known about its evolutionary history and circulating genomic variants, especially in non-privileged countries. The design weaknesses of available MOCV detection/genotyping assays surfaced with recent accumulation of abundant sequence information: all existing MOCV assays fail at accurate genotyping and capturing sub-genotype level diversity. Because complete MOCV genome characterization is an expensive and labor-intensive task, it makes sense to prioritize samples for whole-genome sequencing by diversity triage screening. To meet this demand, we developed a novel assay for accurate MOCV detection and genotyping, and comprehensive sub-genotype qualification to the level of phylogenetic groups (PGs). The assay included a novel set of oligonucleotide primers and probes, and it was implemented using digital polymerase chain reaction (dPCR). It offers sensitive, specific, and accurate detection, genotyping (MOCV1-MOCV3), and PG qualification (PG1-6) of MOCV DNA from clinical samples. The novel dPCR assay is suitable for MOCV diversity triage screening and prioritization of samples for complete MOCV genome characterization.