Avaliação in silico de oligonucleotídeos iniciadores e sondas de PCR para detecção de coronavírus da síndrome respiratória aguda grave 2 e protoparvovírus de carnívoros

Abstract

Primer-target mismatches are a problem that has been approach by researchers in molecular biology, as it is an interference in primer annealing that can prevent the extension step by DNA polymerase. The viruses that will be discussed here have high SNP frequencies, which can cause primer-target mismatches. SARS-CoV-2 is routinely detected by PCR worldwide. The primers and probe sequences to SARS-CoV-2 detection assays were designed according to the first viral sequences available in early 2020, not including the variants that are currently circulate. Canine parvovirus type 2 (CPV-2) is one of the most relevant gastroenteric diseases in small animal medicine and has a high number of SNPs, even though it is a DNA virus. Viral detection can be performed by snap tests, however, the gold standard for CPV-2 detection is PCR. Therefore, different assays have been described and widely used for the molecular detection of CPV-2 in clinical samples. We used SCREENED software to analyze in silico 12 sets of SARS-CoV-2 detection primers in 3,123 genomes of the Gamma, Delta and Omicron variants detected in Brazil. Likewise, we used SCREENED to analyze in silico 7 sets of primers that detect CPV-2, in 996 genomes, of which 28 were vaccine strains widely used worldwide. Overall, the results for both viruses showed that, although most assays continue to detect positive cases and have high coverage and specificity, some mismatches may already result in false negatives or viral quantification problems. [resumo fornecido pelo autor]