Avaliação de iniciadores e protocolo para o diagnóstico da pancitopenia tropical canina por PCR

Resumo

The study was conducted in an attempt to evaluate a protocol and to select primers for the specific diagnosis of Ehrlichia canis by a single-step PCR assay. Sequences of the 16S rRNA gene of the species E. canis (access number = AF162860), E. ewingii (U96436), E. platys (AF1567844), E. chaffeensis, (U86665), E. phagocytophila genogroup (U02521), E. bovis (AF294789) and E. risticii (M21290) were obtained from the Genbank database and then aligned for primer selection. From a semi-conserved region of the alignment it was selected the E. canis specific forward primer which was designated EBR1 (5’- ctctggctataggaaattg- 3’) and, from a conserved region, it was selected a reverse generic primer named EBR5 (5’-ggagtgcttaacgcgttag-3’). Blood samples were collected from ten dogs that were undergoing acute phase of E. canis infection, confirmed by the demonstration of the rickettsia characteristic intracitoplasmatic morulae within the agranulocitic blood cells. The genomic DNA was extracted from each of the blood samples in order to be used for the assessment of the PCR assay in order to evaluate the protocol and the primer pair (EBR1/EBR5) selected in this study. The PCR reaction was positive for all ten isolates of E. canis, yielding the expected fragments of 765 bp size of the 16 S rRNA gene. The lack of positive results with genomic DNA of Babesia canis, Hepatozoon canis, Haemobartonella sp., Trypanosoma evansi and of uninfected host (Canis familiaris) assured confidence on the specivicity of the method for E. canis discrimination.

Descrição

v. 6, n. 1, p. 49-54, jan./mar. 2005.

Palavras-chave

Citação

ALVES, Luciano Marra; LINHARES, Guido; CHAVES, Nilo Sérgio; MONTEIRO, Letícia; LINHARES, Daniel. Avaliação de iniciadores e protocolo para o diagnóstico da pancitopenia tropical canina por PCR. Ciência Animal Brasileira, Goiânia, v. 6, n.1, jan./mar. 2005. Disponível em: <http://www.revistas.ufg.br/index.php/vet/article/view/344/319>.