Development and Validation of a Quantitative Synthetic Analytical Reference Material for Monkeypox Virus

Mpox is a zoonotic viral disease caused by the human monkeypox virus (hMPXV), a double-stranded DNA virus that belongs to the genus Orthopoxvirus and the family Poxviridae. hMPXV is endemic to several countries in Central and Western Africa and has been increasingly appearing in non-endemic areas. The virus causes a smallpox-like disease in humans and is characterized by two distinct genetic clades: Clade I and Clade II. 

Rapid and accurate detection of this virus during the early stages of infection is essential for containment and timely treatment. While culture-based approaches can be used to detect hMPXV, they are typically time-consuming, labor-intensive, and require BSL-3 facilities. PCR-based methods provide a highly sensitive and rapid alternative screening approach. However, the development and validation of these assays depend on high-quality reference materials. To address this need, ATCC® designed and developed a quantitative synthetic molecular standard for hMPXV that contains specific hMPXV diagnostic biomarkers, including those characteristic of both clades. Here, we used a proprietary strategy to incorporate gene sequences typically targeted in various assays for viral detection to create a safe (BSL-1) and reliable positive analytical reference material (ARM). 

The hMPXV ARM was authenticated via next-generation sequencing and quantified via Droplet Digital PCR (ddPCR; Bio-Rad). The synthetic standard’s functionality was tested via 15 published quantitative PCR (qPCR) assays, including those from the Centers for Disease Control and Prevention (CDC) that are specific for non-variola Orthopoxvirus and generic monkeypox. Serial ten-fold dilutions were used to create standard curves, with DNA concentrations ranging from 5.0 x 10⁵  copies/µL to 5 copies/µL. Within this data set, the hMPXV standard had R² values of 0.975 to 0.999, with slopes ranging from -3.291 to -3.477, displaying high efficiency and amplification. 

The data collectively indicate that the hMPXV synthetic molecular standard is an effectively designed ARM suitable for developing and validating molecular-based detection and quantification assays. Our findings confirm the full compatibility of this novel ARM with 15 qPCR assays. Consequently, this ARM can function as a PCR template control, providing a safe and dependable positive control material for molecular assays employed in diagnostics and surveillance.