Monkeypox Virus Detection Assays according to the CDC Guidelines
The recent monkeypox (mpox) outbreak was declared a public health emergency of international concern by the World Health Organization (WHO). At Invitek Diagnostics, we are dedicated to supporting mpox research and assay development. This article aims to provide a comprehensive guide on establishing an effective testing method for Monkeypox virus (MPXV), specifically targeting the West African and Congo Basin strains. Like other infectious pathogens, MPXV infection is diagnosed by PCR and the Centers for Disease Control and Prevention (CDC) has established a generic real-time PCR assay, including specimen collection, DNA isolation and primer and probe design 1, 2.
Current outbreak raised alarm at a global scale
Mpox is a viral disease caused by MPXV, which belongs to the Orthopoxvirus genus. The disease was first identified in humans in 1970 in the Democratic Republic of Congo (DRC). It is endemic to several countries in Central and West Africa, where it has been a persistent health issue for decades. The recent outbreak of mpox has raised significant global concern after a surge in cases with the new variant clade Ib in the DRC and the spread of the virus to neighboring countries. The current outbreak is particularly alarming due to the emergence of a new clade of the virus, which has shown rapid transmission rates and has been linked to sexual contact. The outbreak has not been limited to Africa. Cases have been reported in various countries across Europe and North America, marking a significant shift from previous outbreaks that were largely confined to endemic regions.
Viral Nucleic Acid Extraction Using RTP Pathogen Kit
The recommended specimens for mpox testing by qPCR are skin lesion materials. This includes swabs of the lesion surface, exudate, or lesion crusts. The RTP® Pathogen Kit is a highly efficient and reliable solution for DNA isolation from various sample types and has been successfully used in MPXV testing and other viruses of the Orthopoxvirus genus as supported by numerous scientific publications 3, 4. For detailed instructions and protocols for viral DNA isolation, please download our Whitepaper on the topic.
The RTP® range of products is based on Invitek Diagnostics’ unique, patent-protected non-chaotropic chemistry. RTP® products greatly simplify the process of DNA/RNA purification from pathogen-containing samples. All reagents required for DNA/RNA preparation – including lysis buffer, proteinase and/or lysozyme and carrier nucleic acid (for enhanced recovery of low amounts of target sequences) – are provided as a lyophilized master mix in a single extraction tube. This reagent set is ready-to-use and stable at room temperature. The extraction process is initiated simply by adding a sample to the one-step lysis lyophilized reagents, which represents up to 60% fewer pipetting steps and pipet tips consumption when compared to classical methods (Fig.1).
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Fig.1 - Comparison of classical spin column extraction method with the RTP technology. Due to a 1-step lysis, hands on time and pipetting steps are reduced significantly during lysis while ensuring highest reproducibility
Primers & Probes Specific for MPXV Clades I and II
The CDC has developed several assays for detecting mpox, focusing primarily on real-time PCR testing in clinical specimens, such as swabs from lesions. The CDC’s real-time PCR test procedure, published in June 2022, provides detailed instructions for laboratories, including sequence information for primer and probe development, as well as cycling conditions 1,2.
The assays can differentiate between the various clades of the mpox virus, specifically between Clade I (Congo Basin) and Clade II (West African), which includes Clade IIa and Clade IIb 2. This differentiation is important for understanding the epidemiology of the virus and tailoring public health responses accordingly.
Invitek Diagnostics provides a custom oligo synthesis service specifically for mpox CDC assays, allowing laboratories to acquire high-quality RUO primers and probes tailored to their MPXV testing requirements. Our advanced facilities and rigorous quality control protocols ensure the delivery of reliable and reproducible oligonucleotides.
Ordering process:
- Submit the CDC primer and probe sequence information to our online ordering platform.
- Select the desired purification and scale options.
- Choose the probe dyes according to your multiplexing and instrument requirements.
- Place your order and receive your custom oligos in the chosen formulation.
Be prepared
Establishing a reliable MPXV testing method is essential for public health preparedness and response. Invitek Diagnostics provides specialized support to laboratories by offering high-quality DNA isolation kits, custom oligo synthesis services, and expert technical assistance. By following the guidelines outlined in this article and leveraging our expertise, laboratories can effectively contribute to the accurate detection and control of MPXV.
Additional Mpox Resources:
CDC – Guidance for laboratory personnel.
ECDC – Resources for healthcare professionals.
WHO – Updates on the mpox outbreak and health topics.
References
1 – Centers for Disease Control & Prevention Poxvirus & Rabies Branch (PRB). Test procedure: monkeypox virus generic real‐time PCR test. CDC‐007‐00217. Accessed April 23, 2023. https://www.cdc.gov/poxvirus/monkeypox/pdf/pcr-diagnostic-protocol-508.pdf
2 – Li Y, Zhao H, Wilkins K, Hughes C, Damon IK. Real‐time PCR assays for the specific detection of monkeypox virus West African and Congo Basin strain DNA. J Virol Methods. 2010;169(1):223‐227. doi:10.1016/j.jviromet.2010.07.012
3 – Dumont C, Irenge L M , Magazani E K, Garin D, Muyembe J-JT, et al. Simple Technique for infield Samples Collection in the Cases of Skin Rash Illness and Subsequent PCR Detection of Orthopox viruses and Varicella Zoster Virus. PLoS ONE. 2014;9(5):e96930. doi:10.1371/journal.pone.0096930
4 – Peres MG, Bacchiega TS, Appolinário CM, Vicente AF, Mioni MSR, Ribeiro BLD, Fonseca CRS, Pelícia VC, Ferreira F, Abrahão JS, Megid J. Vaccinia virus in Feces and Urine of Wild Rodents from São Paulo State, Brazil. Viruses. 2018;10(2):51. doi: 10.3390/v10020051.
