Clade-Specific MPXV PCR Assays

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In an evolving infectious disease outbreak, there are two priorities: rapid and accurate detection of the causative agent and characterization of its spread. The polymerase chain reaction (PCR) assay is an effective and portable diagnostic method that can quickly provide information associated with virulence, transmissibility, and pathogenicity. Compared to genomic sequencing, PCR requires less infrastructure, funding, and training. However, the development of sensitive and specific primer sequences is costly, particularly those with subspecies resolution. The recent mpox (monkeypox) virus outbreak underscores the need for the rapid development of clade-specific primers, particularly when there are differences in morbidity and mortality rates. Current mpox assays use primer sequences that also bind to the broader Orthopoxvirus genus, resulting in suspect diagnoses, delays in treatment, and poor allocation of scarce healthcare resources. Additionally, these orthopox-based primer sets cannot distinguish between different mpox clades and cannot illuminate intra-clade evolution over the course of the outbreak. Here, we present the in silico design and in vitro testing of novel clade-specific mpox assays.


There is an ongoing global outbreak of Mpox disease, an illness with a characteristic blistering rash progression. The Mpox virus clusters into two clades with differing levels of virulence and mortality rates. Thus, proper sample identification is critical for surveillance and the public health response programs that rely on such data. Accordingly, the US government specifically lists Clade I under the Federal Select Agent Program. Current diagnostics may fail to identify the virus or its clade membership as the genome mutates. In this work, we demonstrate an end-to-end workflow to quickly evaluate existing PCR assays and design new ones for clade-based identification with respect to large sequence databases in service of preventing a catastrophic fog-of-war from forming during an outbreak.

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