An in silico analysis of PCR-based monkeypox virus detection assays: a
case study for ongoing clinical surveillance
Abstract
The 2022 global mpox outbreak swiftly introduced unforeseen diversity in
the monkeypox virus (MPXV) population resulting in numerous Clade IIb
sublineages. This propagation of new MPXV mutations warrants thorough
re-investigation of previously recommended or validated primers designed
to target MPXV genomes. In this study, we explored 18 PCR primer sets
and examined their binding specificity against 5,210 MPXV genomes,
representing all established MPXV lineages. Our results indicated that
only five primer sets resulted in almost all perfect matches against the
targeted MPXV lineages, and the remaining primer sets all contained 1-2
mismatches against almost all MPXV lineages. We further investigated
mismatch primer-genome pairs and revealed that some of the primers
overlaid with poorly sequenced and assembled regions of the MPXV genomes
that are consistent across multiple lineages. However, we identified 173
99% genome-wide conserved regions across all 5,210 MPXV genomes
representing 30 lineages/clades with at least 80% lineage-specific
consensus for future primer development and primer binding evaluation.
This exercise is crucial to ensure current detection schemes are robust
and serves as a framework for primer evaluation in clinical testing
development for other infectious diseases.