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Development and Validation of a Quantitative Synthetic Molecular Standard for Monkeypox virus

Monkeypox virus

ASM Microbe 2023

Houston, Texas, United States

June 16, 2023


Mpox is a zoonotic viral disease caused by 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. Infections in the current outbreak are from Clade II, which has a low case fatality rate as compared to Clade I. 

Rapid and accurate detection of this virus during the early stages of infection is essential for containment and timely and proper 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 biomarkers for both clades and can serve as a safe and reliable positive control material for diagnostic assays. In addition, this synthetic sequence can serve as a PCR template control for 14 published assays. Here, we used a proprietary strategy to incorporate genes typically targeted in various assays for viral detection and identification. 

As a proof-of-concept, the hMPXV standard was quantified via Droplet Digital™ PCR (ddPCR™; Bio-Rad) and its functionality was tested via published quantitative PCR (qPCR) assays. The synthetic standard was further tested using the Centers for Disease Control and Prevention’s (CDC) non-variola Orthopoxvirus and generic mpox assays. Here, serial ten-fold dilutions were used to create standard curves, with DNA concentrations ranging from 5 copies/µL to 5.0 × 105 copies/µL. Within this data set, the hMPXV standard had R2 values of 0.975 to 0.999 with slopes ranging from M = -3.291 to -3.477, displaying high efficiency and amplification. Overall, the data demonstrate that the hMPXV synthetic molecular standard is a well-designed control that can be used in the development and validation of molecular-based detection and quantification assays. 


Download the poster to explore the use of the Monkeypox virus molecular standard as a control in assay development


Watch the poster presentation


Britany Tang, headshot.

Britany Tang, BS

Senior Biologist, Microbiology R&D, ATCC

Britany Tang is a Senior Biologist at ATCC Microbiology Research and Development. She has extensive experience in the fields of molecular biology and microbiology. Her current work focuses on designing synthetic DNA and RNA molecular standards for molecular diagnostic assays and the development of mock microbial communities for microbiome research. Prior to joining ATCC, her research was centered on arboviral replication and pathogenesis. Britany holds a bachelor’s degree in Biology from George Mason University.

Medical illustration of monbkeypox virus in light blue.

Monkeypox virus molecular diagnostics development

Accurate and rapid diagnosis of mpox is critical for timely healthcare and tracking of transmission. To support the need for increased monitoring, ATCC provides a variety of orthopoxviruses and nucleic acids that support assay development and validation. We have also developed a research use only (RUO) quantitative synthetic monkeypox virus DNA preparation with a sequence design based on several published assays.

Develop your mpox assay