Author(s): Tozato, Claudia de Camargo [UNESP] ; Zadra, Vívian Ferreira [UNESP] ; Basso, Caroline Rodrigues [UNESP] ; Araújo Junior, João Pessoa [UNESP]
Date: 2018
Persistent ID: http://hdl.handle.net/11449/173188
Origin: Oasisbr
Subject(s): Canine distemper; Centrifugation; Molecular diagnosis; One-Step RT-qPCR; Urine
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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 and 10−2 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.
Departamento de Microbiologia e Imunologia Instituto de Biociências Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)
Departamento de Química e Bioquímica Instituto de Biociências Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)
Instituto de Biotecnologia (IBTEC) Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)
Departamento de Microbiologia e Imunologia Instituto de Biociências Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)
Departamento de Química e Bioquímica Instituto de Biociências Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)
Instituto de Biotecnologia (IBTEC) Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)
