Mutations in Animal SARS-CoV-2 Induce Mismatches with the Diagnostic PCR Assays

Abstract

Recently, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was detected in several animal species. After transmission to animals, the virus accumulates mutations in its genome as adaptation to the new animal host progresses. Therefore, we investigated whether these mutations result in mismatches with the diagnostic PCR assays and suggested proper modifications to the oligo sequences accordingly. A comprehensive bioinformatic analysis was conducted using 28 diagnostic PCR assays and 793 publicly available SARS-CoV-2 genomes isolated from animals. Sixteen out of the investigated 28 PCR assays displayed at least one mismatch with their targets at the 0.5% threshold. Mismatches were detected in seven, two, two, and six assays targeting the ORF1ab, spike, envelope, and nucleocapsid genes, respectively. Several of these mismatches, such as the deletions and mismatches at the 3’ end of the primer or probe, are expected to negatively affect the diagnostic PCR assays resulting in false-negative results. The modifications to the oligo sequences should result in stronger template binding by the oligos, better sensitivity of the assays, and higher confidence in the result. It is necessary to monitor the targets of diagnostic PCR assays for any future mutations that may occur as the virus continues to evolve in animals.

Comparison of analytical sensitivity and efficiency for SARS-CoV-2 primer sets by TaqMan-based and SYBR Green-based RT-qPCR

“…nCoV_IP2 and ORF1ab both contained only one primer or probe with only one significant mismatch with only one variant ( SI Table S12 - 15 ). The NIID-2019-nCoV_2 reverse primer showed a single-nucleotide mismatch (G-C) with all tested sequences ( SI Table 12 – 16 ) as reported previously (Elaswad and Fawzy 2021 ). However, if this could be corrected, the bespoke reverse primer would only have one slightly significant mismatch (C-T) for Lambda variants.…”

“…Though mismatches of ORF1ab was found in animal isolates (Elaswad and Fawzy 2021 ), up to now, it is still almost perfect for human ones according to previous studies (Alvarez-Diaz et al 2020 ; Khan and Cheung 2020b ) and our results ( SI Table 12 – 16 ). In fact, ORF1ab was recommended as the most sensitive and reliable one repeatedly (Jung et al 2020 ; Mollaei et al 2020 ).…”

“…Their low sensitivity may be explained by the presence of multiple wobble nucleotides (Eis-Hubinger et al 2020 ; Toptan et al 2020 ). Moreover, as found in this study ( SI Table 12 & 14 ) and several previous studies (Alvarez-Diaz et al 2020 ; Elaswad and Fawzy 2021 ; Khan and Cheung 2020b ; Miranda and Weber 2021 ; Pillonel et al 2020 ), they did not fully match the available SARS-CoV-2 genomes. Though the mismatches had little effect on their efficiencies (Corman and Drosten 2020 ; Nalla et al 2020 ) and increased their coverage, they also increased the coverage for SARS-CoV-1 and other human coronaviruses (Miranda and Weber 2021 ).…”

“…In previous studies, among our choices, nCoV_IP4 was the only one targeting the ORF1 gene which matched perfectly with animal and human isolates (Elaswad and Fawzy 2021 ; Khan and Cheung 2020b ). However, we found it imperfect for Delta, Kappa, and Mu variants ( SI Table 12 , 14 , 16 ).…”

“…All primer–probe sets have at least one significant detected mismatch frequency which means above the 0.5% threshold (Elaswad and Fawzy 2021 ; Khan and Cheung 2020b ; Vogels et al 2020 ) with all six variants ( SI Table S12 - 16 ). nCoV_IP2 and ORF1ab both contained only one primer or probe with only one significant mismatch with only one variant ( SI Table S12 - 15 ).…”