Development of an ARMS-Quadruplex-qPCR assay for the rapid identification of MPXV and the clades Ia, Ib, IIa and IIb

Monkeypox was re-emerging in 2022 and spread to more than 100 countries. Two clades of Monkeypox virus (MPXV) result in different lethality rates and varying transmission capabilities. Rapid identification of MPXV and differentiation of its clades and subclades are crucial for effective control of the disease. In this study, we developed an ARMS-Quadruplex-qPCR method to detect MPXV and distinguish clades (Ia, Ib, IIa and IIb). F3L gene was used to detect all clades of MPXV from other orthopoxviruses. A 1953 bp fragment containing the C3L gene was found to be completely absent in clade II. Additionally, a sequence spanning from the 177th to the 1318th position (1142 bp) within the 1953 bp fragment was missing in Ib. Therefore, the 1142 bp sequence was used to distinguish Ia from other subclades, and the sequence with the 1142 bp region missing in Ib was used to discriminate Ib from other subclades. Since subclades IIa and IIb are too close to have large deletions and insertions, a unique single nucleotide polymorphism (SNP) was used to design a primer/probe set for ARMS-qPCR to differentiate clade IIa from IIb. The ARMS-Quadruplex-qPCR system can detect down to 2 copies per reaction of MPXV and effectively differentiate all the four subclades. Altogether, four qPCR primer/probe sets in one tube were deployed to recognize MPXV and differentiate MPXV subclades. The high sensitivity, rapidity and specificity of the developed system make it a promising alternative for the diagnosis of MPXV and the determination of the subclades of the infected MPXV.