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

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.