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Ciência Rural

versão impressa ISSN 0103-8478versão On-line ISSN 1678-4596

Resumo

TOZATO, Claudia de Camargo; ZADRA, Vívian Ferreira; BASSO, Caroline Rodrigues  e  ARAUJO JUNIOR, João Pessoa. Canine distemper virus detection by different methods of One-Step RT-qPCR. Cienc. Rural [online]. 2016, vol.46, n.9, pp.1601-1606.  Epub 17-Maio-2016. ISSN 0103-8478.  https://doi.org/10.1590/0103-8478cr20150932.

Three commercial kits of One-Step RT-qPCR were evaluated for the molecular diagnosis of Canine Distemper Virus. Using the kit that showed better performance, two systems of Real-time RT-PCR (RT-qPCR) assays were tested and compared for analytical sensitivity to Canine Distemper Virus RNA detection: a One-Step RT-qPCR (system A) and a One-Step RT-qPCR combined with NESTED-qPCR (system B). Limits of detection for both systems were determined using a serial dilution of Canine Distemper Virus synthetic RNA or a positive urine sample. In addition, the same urine sample was tested using samples with prior centrifugation or ultracentrifugation. Commercial kits of One-Step RT-qPCR assays detected canine distemper virus RNA in 10 (100%) urine samples from symptomatic animals tested. The One-Step RT-qPCR kit that showed better results was used to evaluate the analytical sensitivity of the A and B systems. Limit of detection using synthetic RNA for the system A was 11 RNA copies µL-1 and 110 RNA copies µl-1 for first round System B. The second round of the NESTED-qPCR for System B had a limit of detection of 11 copies µl-1. Relationship between Ct values and RNA concentration was linear. The RNA extracted from the urine dilutions was detected in dilutions of 10-3 and10-2 by System A and B respectively. Urine centrifugation increased the analytical sensitivity of the test and proved to be useful for routine diagnostics. The One-Step RT-qPCR is a fast, sensitive and specific method for canine distemper routine diagnosis and research projects that require sensitive and quantitative methodology.

Palavras-chave : canine distemper; molecular diagnosis; One-Step RT-qPCR; urine; centrifugation.

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