Diversidade nucleotídica do gene N do núcleocapsídeo do vírus da cinomose (CDV) e o impacto em sequências de oligonucleotídeos e sondas de RT-qPCR.

Abstract

Canine distemper virus (CDV) is a highly relevant infectious agent in animal health, causing outbreaks with high morbidity and mortality, especially in unvaccinated dogs. RT-qPCR (Reverse Transcriptase - quantitative Polymerase Chain Reaction) is the reference technique for the accurate diagnosis of distemper, and the choice of primers and probes is crucial for an accurate diagnosis. This study aimed to evaluate the nucleotide diversity of the nucleocapsid (N) gene of CDV in the target regions of the primers and probe used in RT-qPCR. For this purpose, a survey of 62 complete and incomplete sequences of the CDV N gene was performed in the GenBank database, followed by a multiple alignment of the sequences obtained from GenBank together with the primers and probe from the literature, to verify the existence or not of nucleotide diversity. The results demonstrated the presence of nucleotide diversity in the target regions of the primers and probe described in the literature, which may compromise the specificity of the reaction. In view of this, new primers and probe were designed in a conserved region of the N gene, aiming at greater specificity and a lower risk of amplification of nonspecific sequences. The in-silico validation of the new primers and probe indicated high specificity for the detection of the distemper virus. It is concluded that the genetic diversity of CDV directly impacts the efficacy of the primers and probe used in RT-qPCR. The development of new primers and probe, based on conserved regions of the N gene, represents a significant advance for the molecular diagnosis of canine distemper virus, ensuring greater accuracy and reliability of the results.