An In Silico Analysis of PCR-Based Monkeypox Virus Detection Assays: A Case Study for Ongoing Clinical Surveillance

• Published in: Viruses Vol. 15; no. 12; p. 2327
• Main Authors: Song, Kuncheng, Brochu, Hayden N, Zhang, Qimin, Williams, Jonathan D, Iyer, Lakshmanan K
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 27-11-2023
• Subjects: Binding sites; Biological Assay; Case studies; Consensus; Design; Diagnosis; Disease Outbreaks; Genomes; Human monkeypox; Humans; in silico analysis; Infectious diseases; Medical tests; Monkeypox; monkeypox virus; Monkeypox virus - genetics; Mutation; Pathogens; Polymerase Chain Reaction; poxvirus; Public health; quantitative polymerase chain reaction; Virus research; Viruses; United States; in silico analysis; quantitative polymerase chain reaction; poxvirus; monkeypox virus; monkeypox
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v15122327

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 the 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 5210 MPXV genomes, representing all the 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 the MPXV lineages. We further investigated the mismatched primer-genome pairs and discovered that some of the primers overlapped with poorly sequenced and assembled regions of the MPXV genomes, which are consistent across multiple lineages. However, we identified 173 99% genome-wide conserved regions across all 5210 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 that the current detection schemes are robust and serve as a framework for primer evaluation in clinical testing development for other infectious diseases.