In a recent study posted to the medRxiv* preprint server, researchers analyzed the primer and probe sequences of america Centers for Disease Control and Prevention (CDC) advisable monkeypox virus (MPXV) generic real-time polymerase chain response (PCR) assay(s).

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

Background

MPXV, a species of the genus orthopoxvirus, has a double-stranded deoxyribonucleic acid (DNA) genome. The World Health Organization (WHO) declared MPXV re-emergence a public health emergency on July 23, 2022, following reports of over 66,000 cases in 106 countries. MPXV is the second such virus that’s spreading rapidly worldwide after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) steadily became an endemic virus.

As for SARS-CoV-2, the CDC released a real-time PCR test for MPXV detection in wastewater samples that deployed generic primers and probes targeting a West African and a Congo Basin MPXV strain. Likewise, other real-time PCR assays detect MPXV in clinical samples.

The difficulty is that the primers and probes utilized in these generic PCR assays are based on MPXV strains circulating almost a decade ago between 2002 and 2009. Given the rapid rate at which all DNA viruses evolve, the regions targeted by those oligos used for MPXV detection will need to have undergone substantial mutations.

Concerning the study

In the current study, researchers retrieved 683 MPXV complete genomes from the worldwide initiative on avian influenza data (GISAID) database, available till August 5, 2022, and aligned their oligo sequences against the primer sequences of the currently used MPXV diagnostic assays. Further, they aligned primer and probe sequences and their reverse complements within the seven real-time PCR assays against 1,779 MPXV genomes and computed the share of genomes with a 100% match. These assays targeted O2L, F3L, C3L, and G2R genes for MPXV detection.

The team also evaluated three real-time PCR assays detecting viruses of the Orthopoxvirus genus. Finally, the researchers synthesized the mismatch-corrected primers or an artificial MPXV gene fragment to check the mismatch effects and compared them to the generic assay for MPXV detection.

Study findings

They used 1,730 complete MPXV genomes retrieved in the course of the 2022 worldwide outbreak. Aligning the three oligo sequences and their reverse complements from the MPXV generic assays yielded MPXV generic forward sequences (MPXV-F) with 100% identity to 4 genomes and generic reverse primer (MPXV-R) matching to 1.73% of the genomes. Further, the researchers found 31 sequence mismatches in 99.08% and 97.46% of MPXV generic forward sequences (MPXV-F) and 32 generic reverse primers (MPXV-R), respectively.

While the previous had a single synonymous mutation A194165G, in 99.08% of the published MPXV genomes, the latter had a non-synonymous substitution, G194233A, in 97.46% of the genomes. Quite the opposite, the MPXV probe sequence (MPV-P) was conserved and matched 99.31% of the genomes within the database.

The first study finding was that the currently used MPXV generic assays won’t be optimally and accurately detecting MPXV. No matter its position, every mismatch throughout the primer sequence could reduce the thermal stability of the primer-template DNA duplex and impact PCR performance. The authors noted a two primer-template mismatch, which had combined effects on PCR performance. It yielded a ~11-fold poorer estimation of the initial template DNA and a four-fold increase within the 95% limit of detection (LOD). Hence, the mismatch-corrected assay with absolute complementarity between primers and current MPXV genomes could provide a more sensitive and accurate MPXV detection.

Further,  the study results showed that genetic variations within the primer-probe regions of MPXV genomes could indicate a temporal and spatial emergence pattern of monkeypox disease. Three assays, MPV_F3L, MPV_G2R_WA, 283, and OPV_F8L, showed the best matching rating of over >99% to the worldwide MPXV genome database in 2022. Nonetheless, the alternative of the assay also varies with the sample type. As an example, these three assays worked well for clinical samples but not wastewater samples, which could contain human waste and that of cats, dogs, mice, rabbits, and cows.

Conclusion

The mismatch-corrected assay developed within the study had over 97% complementarity to MPXV genomes. Thus, it showed higher sensitivity and improved quantification potential and will aid the MPXV detection.

Improved MPXV diagnostic capabilities are crucial as public health officers and clinicians fight the MPXV outbreak. Thus, future studies should concentrate on developing an excellent higher MPXV diagnostic assay that accounts for other aspects that impact the diagnostic efficiency and mismatch-induced effects (e.g., the standard of the template DNA, the PCR master mix, and primer dimer formation).

*Essential notice

medRxiv publishes preliminary scientific reports that should not peer-reviewed and, due to this fact, mustn’t be considered conclusive, guide clinical practice/health-related behavior, or treated as established information.