Wide mismatches in the sequences of primers and probes for monkeypox virus diagnostic assays

Abstract

Rapid and accurate diagnosis of infections is fundamental to containment of disease. Several monkeypox virus (MPV) real‐time diagnostic assays have been recommended by the CDC; however, the specificity of the primers and probes in these assays for the ongoing MPV outbreak has not been investigated. We analyzed the primer and probe sequences present in the CDC recommended MPV generic real‐time PCR assay by aligning those sequences against 1730 MPV complete genomes reported in 2022 worldwide. Sequence mismatches were found in 99.08% and 97.46% of genomes for the MPV generic forward and reverse primers, respectively. Mismatch‐corrected primers were synthetized and compared to the generic assay for MPV detection. Results showed that the two primer‐template mismatches resulted in a ~11‐fold underestimation of initial template DNA in the reaction and 4‐fold increase in the 95% LOD. We further evaluated the specificity of seven other real‐time PCR assays used for MPV and orthopoxvirus (OPV) detection and identified two assays with the highest matching score (>99.6%) to the global MPV genome database in 2022. Genetic variations in the primer‐probe regions across MPV genomes could indicate the temporal and spatial emergence pattern of monkeypox disease. Our results show that the current MPV real‐time generic assay may not be optimal to accurately detect MPV, and the mismatch‐corrected assay with full complementarity between primers and current MPV genomes could provide a more sensitive and accurate detection of MPV.

Implementation and clinical evaluation of an Mpox virus laboratory‐developed test on a fully automated random‐access platform

“…Notably, MPXV isolates of the current outbreak exhibit mismatches in the primer binding sites of both the forward and reverse primer 14 . A recent report indicates a fourfold increase of the LoD of the PCR described by Li et al 10 on the current MPXV subvariants 15 . Since other mutations in the MPXV genome might further increase the LoD of this PCR, we recommend following current guidelines of local health authorities.…”

“…14 A recent report indicates a fourfold increase of the LoD of the PCR described by Li et al 10 on the current MPXV subvariants. 15 we recommend following current guidelines of local health authorities.…”

A Multi-Laboratory Evaluation of Commercial Monkeypox Virus Molecular Tests

“…In order to perform a robust and informative assay, we combined the general (GE) and WA clade-specific (WA) reactions described by Li et al (2010) ( 7 ) with an internal control reaction and demonstrated that the 2 MPXV reactions are sensitive and have very similar kinetics. A recent publication ( 15 ) suggested that changes in primers sequences developed by Li et al ( 7 ) should be made, to render them more suitable to detect the currently circulating MPXV clade. In our reaction design, we addressed this by altering the sequence of the MPXV general (GE) reaction forward primer, already in May 2022, based on genomic analysis of currently circulating MPXV isolates.…”

Rapid identification of full-length genome and tracing variations of monkeypox virus in clinical specimens based on mNGS and amplicon sequencing

“…Besides, Wu et al. found variations in the target regions of the monkeypox virus detection primers published by the CDC ( Wu F. et al., 2023 ). These newly emerging variations could lead to an underestimation of the viral load in samples and even result in misdiagnosis.…”