Padronização de um teste de diagnóstico para Estomatite Vesicular por PCR em tempo real

Abstract

Vesicular stomatitis was first described in the United States in 1926 as a vesicular disease of horses, and subsequently of cattle and swine. The disease is characterized primarily by formation of vesicles on the tongue, lips, oral mucosa, teats and the coronary band of the animals feet. The vesicular stomatitis virus (VSV), the disease causative agent, its an enveloped negative strand RNA virus that belongs to Vesiculovirus genus, family Rhabdoviridae. Vesicular stomatitis is clinically indistinguishable from other vesicular diseases, like foot and mouth disease, swine vesicular exanthema and swine vesicular disease, when equines are not involved. This brings great socio-economic implications and in the absence of a clinical diagnosis, laboratory diagnosis becomes urgent and indispensable in case of suspicion of vesicular stomatitis. In this work, the genetic material of a VSV reference sample was used as template in conventional PCR to obtain a plasmid positive control of the reaction. The reference sample was characterized from the plasmid sequencing with VSV insert. For qPCR, primers were designed in a conserved region of the L gene (viral RNA polymerase), from the reference strain (VSIV) sequences and field samples (VSAV and COCV). The standardization of reaction with plasmid control was made with homemade mix, and the final concentration of each primer was 1,25 mol/100L, with 10 M of dNTP and 2,0 mM of MgCl2. The dyes used were Evagreen for detection system and ROX® as passive reference. The reaction was optimized to increase the sensitivity and the final parameters were as follows: 4,0 mol/100L of each primer, 100 M of dNTP and 2,0 mM of MgCl2. Genes of -actin and HPRT were chosen to be used as internal controls of reaction. The assay presented satisfactory results when using the commercial mix, but in need of improvements especially regard with primers for use in the diagnosis of bovine vesicular stomatitis.