Development of a multiplex real-time PCR assay for the simultaneous detection of mpox virus and orthopoxvirus infections

Since May 2022, the multi-country outbreak of monkeypox (mpox) has raised a great concern worldwide. Early detection of mpox virus infection is recognized as an efficient way to prevent mpox transmission. Mpox specific detection methods reported up to now are based on the SNPs among mpox virus and other orthopoxviruses. We have therefore developed a real-time PCR based mpox detection method targeting mpox virus specific sequences (N3R and B18Rplus). We have also optimized an orthopoxvirus detection system which targets the highly conserved E9L and D6R genes. The mpox and orthopoxvirus real-time PCR assays have a high sensitivity (1 copy/reaction) and specificity. Mpox viral DNA and clinical samples from mpox patients are detected with the mpox detection system. Furthermore, we have established a multiplex real-time PCR detection system allowing simultaneous and efficient detection of mpox and orthopoxvirus infections. •A new mpox detection method based on mpox virus specific sequences.•The mpox and orthopoxvirus real-time PCR assays have a high sensitivity (1 copy/reaction).•New multiplex real-time PCR system allows simultaneous and efficient detection of mpox and orthopoxvirus infections.