Novo PCR em tempo real sensível que visa o gene p28 para a detecção de Ehrlichia canis em amostras de sangue de cães
2021
This study aims to describe a new detection method of a quantitative real-time polymerase chain reaction (qPCR) targeting the
28 kDa outer membrane protein gene (p28) as well as to compare this method with a conventional PCR (cPCR), which targets the same gene,
in order to evaluate the performance of the technique designed in this study in detecting Ehrlichia canis (E. canis). Optimum oligonucleotides
concentrations were reached, and the analytical sensitivity and specificity of the qPCR were performed. A total of 218 dogs’ whole blood
samples were conventionally collected for this study. The DNA was extracted from each sample. Subsequently, the samples were tested by
an established cPCR and the new qPCR to compare each technique’s performances. This new qPCR method for the molecular detection of
E. canis presented a detection limit of ten copies of the fragment and was considered specific for E. canis according to analytical specificity
analyses performed in vitro and in silico. The standard curve revealed 100% efficiency and a coefficient of determination (R2) equivalent to
99.8%. Among the samples examined by qPCR, 24.31% were considered positive, significantly greater than those detected by cPCR (15.13%).
The qPCR technique reached a higher sensitivity than the cPCR when targeting the p28 gene in detecting E. canis. The qPCR standardized in
this study is an efficient method for confirming canine monocytic ehrlichiosis (CME) diagnosis and might provide the parasitemia monitoring
during the disease treatment.
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