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Mismatches in the primer and probe sequences of current monkeypox virus diagnostic tests need to be corrected to improve detection accuracy
In a recent study published on the medRxiv* preprint server, researchers analyzed the primer and probe sequences of the generic real-time polymerase chain reaction (PCR) tests for monkeypox virus (MPXV) recommended by the United States Centers for Disease Control and Prevention (CDC). Study: Large mismatches in the sequences of primers and probes for monkeypox virus diagnostic tests. Photo credit: Dotted Yeti/Shutterstock This news article was a review of a preliminary scientific report that had not been peer-reviewed at the time of publication. Since its initial publication, the scientific report has now been peer-reviewed and accepted for publication in an academic journal. Links to the…
Mismatches in the primer and probe sequences of current monkeypox virus diagnostic tests need to be corrected to improve detection accuracy
In a recent study published in medRxiv * Preprint Server, Researchers analyzed the primer and probe sequences of the United States Centers for Disease Control and Prevention (CDC) recommended generic real-time polymerase chain reaction (PCR) tests for monkeypox virus (MPXV).
Studie: Große Fehlpaarungen in den Sequenzen von Primern und Sonden für Diagnosetests für das Monkeypox-Virus. Bildnachweis: Dotted Yeti/Shutterstock
This news article was a review of a preliminary scientific report that had not been peer-reviewed at the time of publication. Since its initial publication, the scientific report has now been peer-reviewed and accepted for publication in an academic journal. Links to the preliminary and peer-reviewed reports can be found in the Sources section at the end of this article. View sources
background
MPXV, a species of the genus Orthopoxvirus, has a double-stranded deoxyribonucleic acid (DNA) genome. The World Health Organization (WHO) declared the resurgence of MPXV a public health emergency on July 23, 2022, after over 66,000 cases were reported in 106 countries. MPXV is the second such virus to spread rapidly worldwide after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gradually became an endemic virus.
Regarding SARS-CoV-2, the CDC published a real-time PCR test for MPXV detection in wastewater samples using 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 problem is that the primers and probes used in these generic PCR assays are based on MPXV strains that circulated almost a decade ago between 2002 and 2009. Given the rapid evolution of all DNA viruses, the regions targeted by these oligos used for MPXV detection must have undergone significant mutations.
About the study
In the present study, researchers retrieved 683 complete MPXV genomes from the Global Initiative on Avian Influenza Data (GISAID) database, available until August 5, 2022, and aligned their oligo sequences with the primer sequences of currently used MPXV diagnostic tests. In addition, they aligned primer and probe sequences and their reverse complements in the seven real-time PCR assays to 1,779 MPXV genomes and calculated the percentage of genomes with 100% agreement. These assays targeted the O2L, F3L, C3L, and G2R genes for MPXV detection.
The team also evaluated three real-time PCR assays for the detection of viruses from the Orthopoxvirus genus. Finally, the researchers synthesized the mismatch-corrected primers or a synthetic MPXV gene fragment to test the mismatch effects and compared them with the generic assay for MPXV detection.
Study results
They used 1,730 complete MPXV genomes sampled during the 2022 global outbreak. Alignment of the three oligo sequences and their reverse complements from the generic MPXV assays yielded generic MPXV forward sequences (MPXV-F) with 100% identity to four genomes and generic reverse primers (MPXV-R) that matched 1.73% of the genomes. In addition, 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 former had a single synonymous mutation, A194165G, in 99.08% of published MPXV genomes, the latter had a nonsynonymous substitution, G194233A, in 97.46% of genomes. On the contrary, the MPXV probe sequence (MPV-P) was conserved and matched to 99.31% of the genomes in the database.
The primary finding of the study was that currently used generic MPXV tests may not optimally and accurately detect MPXV. Regardless of position, any mismatch within the primer sequence could reduce the thermal stability of the primer-template DNA duplex and affect PCR performance. The authors noted a mismatch between two primer templates that had a combined impact on PCR performance. It gave an approximately 11-fold worse estimate of the initial template DNA and a four-fold increase in the limit of detection (LOD) of 95%. Therefore, the mismatch-corrected assay with absolute complementarity between primers and current MPXV genomes could enable more sensitive and accurate MPXV detection.
In addition, the study results showed that genetic variations in the primer-probe regions of the MPXV genome could indicate a temporal and spatial pattern of emergence of monkeypox disease. Three assays, MPV_F3L, MPV_G2R_WA, 283 and OPV_F8L, showed the highest concordance score of over >99% with the global MPXV genome database in 2022. However, the choice of assay also varies depending on the sample type. For example, these three tests worked well for clinical samples, but not for wastewater samples, which could contain human waste as well as feces from cats, dogs, mice, rabbits and cows.
Diploma
The mismatch-corrected assay developed in the study demonstrated over 97% complementarity to MPXV genomes. Thus, it showed higher sensitivity and improved quantification potential and could aid MPXV detection.
Improved MPXV diagnostic capabilities are critical as public health officials and physicians combat the MPXV outbreak. Future studies should therefore focus on developing an even better MPXV diagnostic assay that takes into account other factors that influence diagnostic efficiency and mismatch-related effects (e.g., the quality of template DNA, PCR master mix, and primer-dimer formation).
This news article was a review of a preliminary scientific report that had not been peer-reviewed at the time of publication. Since its initial publication, the scientific report has now been peer-reviewed and accepted for publication in an academic journal. Links to the preliminary and peer-reviewed reports can be found in the Sources section at the end of this article. View sources
References:
- Vorläufiger wissenschaftlicher Bericht.
Fuqing Wu, Jeremiah Oghuan, Anna Gitter, Kristina D. Mena, Eric L. Brown. (2022). Große Fehlpaarungen in den Sequenzen von Primern und Sonden für Diagnosetests für das Monkeypox-Virus. medRxiv. doi: https://doi.org/10.1101/2022.08.10.22278644 https://www.medrxiv.org/content/10.1101/2022.08.10.22278644v2 - Von Experten begutachteter und veröffentlichter wissenschaftlicher Bericht.
Wu, Fuqing, Jeremiah Oghuan, Anna Gitter, Kristina D. Mena und Eric L. Brown. 2022. „Große Fehlpaarungen in den Sequenzen von Primern und Sonden für Monkeypox-Virus-Diagnosetests.“ Journal of Medical Virology 95 (1). https://doi.org/10.1002/jmv.28395. https://onlinelibrary.wiley.com/doi/10.1002/jmv.28395.
Article revisions
- 15. Mai 2023 – Das vorab gedruckte vorläufige Forschungspapier, auf dem dieser Artikel basiert, wurde zur Veröffentlichung in einer von Experten begutachteten wissenschaftlichen Zeitschrift angenommen. Dieser Artikel wurde entsprechend bearbeitet und enthält nun einen Link zum endgültigen, von Experten begutachteten Artikel, der jetzt im Abschnitt „Quellen“ angezeigt wird.