Evaluation and Clinical Validation of Pan-Specific and Clade-Specific Diagnostic Real-Time PCR Assays for Monkeypox Virus.

Abstract

Human mpox, formerly known as monkeypox, has twice been declared a Public Health Emergency of International Concern (PHEIC) by the World Health Organization, in 2022 and 2024. Despite this, global access to rapid, reliable diagnostics remains limited, hindering outbreak control and equitable clinical response. To address this gap, we developed and validated a rapid direct real-time PCR assay capable of detecting and differentiating Clade I and Clade II monkeypox virus (MPXV) directly from clinical samples. We designed a new pan-specific B15L assay to complement the published F3L assay and introduced the first clade-specific assay targeting the B1R gene of Clade I MPXV. In silico validation showed that both B15L and F3L assays detected all available MPXV genomes with 100% sensitivity, while B1R selectively detected only Clade I genomes with 100% specificity. Cross-reactivity screening against 40 high-priority non-target organisms revealed minimal risk of false positives. We confirmed analytical sensitivity of 2 copies per reaction for all assays in vitro, with clade-specific B1R showing 100% specificity. When adapted into a lyophilized direct PCR format for near-point-of-care use, the assay demonstrated detection down to 1 copy per reaction for pan-specific targets and 2 copies for clade-specific detection in spiked clinical samples. This direct PCR assay delivers accurate results in under 1 h without requiring nucleic acid extraction. Our combined genomic, laboratory, and clinical validations demonstrate exceptional sensitivity and specificity, positioning this assay as a highly promising tool in emergency mpox diagnostics and a potential model for future outbreak-responsive testing platforms.