A dual-target real-time PCR for proactive detection of Mpox variants

In 2022, cases of Monkeypox virus (MPXV) in California contained a mutation in the TNF receptor gene (GR2G) that rendered the virus undetectable using a widely adopted public health diagnostic qPCR assay. This underscored the need for a dual-target PCR approach and prompted validation of a second target by the BCCDC Public Health Laboratory. In addition to the GR2G target validated in the original qPCR assay (and duplexed with the endogenous target human β-globin (HBG)), GP113 (OPG128) was identified and validated using both clinical samples and MPXV DNA controls. Mutations in GR2G and GP113 (found in the emerging clade Ib) were also addressed along with the updated target. The new triplex assay (GR2G/GP113/HBG) had 100 % inclusivity and 100 % accuracy with all clinical samples tested and did not cross-react with herpes simplex virus-1 or -2, varicella zoster virus, or enterovirus. It showed < 5 % coefficient of variance between replicates and had a limit-of-detection of 10 copies/μL for GR2G and GP113. The use of two targets presents redundancy against further mutations in MPXV and is recommended for use with all viral qPCR assays moving forward.