Study: Wide mismatches in the sequences of primers and probes for Monkeypox virus diagnostic assays. Image Credit: Dotted Yeti/Shutterstock

Mismatches in primer and probe sequences for current monkeypox virus diagnostic assays require correction to improve detection accuracy

In a recent study published in medRxiv* Prepress server, researchers analyzed primer and probe sequences for the generic PCR assay(s) recommended by the US Centers for Disease Control and Prevention (CDC).

Stady: Extensive mismatch in sequences of primers and probes for monkeypox virus diagnostic assays. Image Credit: Dotted Yeti / Shutterstock


MPXV, a species of the genus orthopoxvirus, has a double deoxyribonucleic acid (DNA) genome. The World Health Organization (WHO) declared the re-emergence of MPXV a public health emergency on July 23, 2022, after reports of more than 66,000 cases in 106 countries. MPXV is the second virus to spread rapidly worldwide after SARS-CoV-2 has gradually become endemic.

For SARS-CoV-2, the CDC released a real-time PCR assay to detect MPXV in wastewater samples that published primers and generic investigations targeting West Africa and the Congo Basin strain MPXV. Similarly, other real-time PCR assays detect MPXV in clinical samples.

The problem is that the primers and probes used in these general PCR assays are based on MPXV strains that have been circulating for nearly a decade between 2002 and 2009. Due to the rapid rate at which all DNA viruses evolve, the regions targeted by those oligos are used to detect MPXV must have undergone significant mutations.

about studying

In this study, researchers retrieved 683 complete MPXV genomes from the Global Initiative for Avian Influenza Database (GISAID) database, available through August 5, 2022, and aligned their oligo sequences to the primer sequences of the currently used MPXV diagnostic assays. Furthermore, they aligned primer and probe sequences and their reverse complements in the seven real-time PCR assays against 1,779 MPXV genomes and calculated the percentage of genomes with a 100% match. These tests targeted the O2L, F3L, C3L, and G2R genes for MPXV detection.

The team also evaluated three real-time PCR assays for the detection of Orthopoxviruses. Finally, the researchers assembled mismatch-corrected primers or a synthetic MPXV gene fragment to test the effects of mismatches and compare them to the general assay for MPXV detection.


They used 1,730 complete MPXV genomes recovered during the worldwide outbreak of 2022. Alignment of the three oligo sequences and their reverse complementation from the MPXV generic assays yielded generic MPXV forward sequences (MPXV-F) with 100% identity for four genomes and a generic reverse primer. (MPXV-R) corresponds to 1.73% of the genomes. Moreover, the researchers found 31 non-identical sequences in 99.08% and 97.46% of the MPXV generic forward (MPXV-F) and 32 general reverse primers (MPXV-R) sequences, respectively.

Whereas the former had a single synonymous mutation A194165G, in 99.08% of published MPXV genomes, the latter had a non-synonymous variant, G194233A, in 97.46% of genomes. Conversely, MPXV (MPV-P) probe sequences were conserved and matched 99.31% of the genomes in the database.

The primary finding of the study was that the general MPXV assays currently in use may not optimally and accurately detect MPXV. Regardless of its location, each mismatch within the primer sequence can reduce the thermal stability of the primer template DNA duplex and affect PCR performance. The authors noted a primer template mismatch, which had combined effects on PCR performance. It resulted in an 11-fold weaker estimation of the template’s primary DNA and a four-fold increase in the 95% limit of detection (LOD). Hence, mismatch-corrected screening of absolute complementarity between primers and current MPXV genomes could provide more sensitive and accurate MPXV detection.

Moreover, the study results showed that genetic differences in the primer probe regions of MPXV genomes could indicate a temporal and spatial onset pattern of monkeypox. Three assays, MPV_F3L, MPV_G2R_WA, 283, and OPV_F8L, showed the highest matching score of over 99% to the global MPXV genome database in 2022. However, the selection of the assay also varies with sample type. For example, these three tests worked well on clinical samples but not sewage samples, which may contain human excreta and those of cats, dogs, mice, rabbits, and cows.


The mismatch-corrected assay developed in the study had more than 97% complementarity with the MPXV genomes. Thus, it has shown higher sensitivity and improved quantification potential and can aid in MPXV detection.

The improved diagnostic capabilities of MPXV are critical as public health officials and clinicians battle MPXV outbreaks. Thus, future studies should focus on developing a better MPXV diagnostic assay that takes into account other factors influencing diagnostic efficiency and the effects caused by mismatches (eg, template DNA quality, PCR master mix, dimer-primer formation).

*Important note

medRxiv publishes preliminary scientific reports that have not been peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health-related behavior, or be treated as established information.

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