| Full text | |
| Author(s): |
Tozato, Claudia de Camargo
;
Zadra, Vivian Ferreira
;
Basso, Caroline Rodrigues
;
Araujo Junior, Joao Pessoa
Total Authors: 4
|
| Document type: | Journal article |
| Source: | Ciência Rural; v. 46, n. 9, p. 1601-1606, SEP 2016. |
| Web of Science Citations: | 0 |
| Abstract | |
ABSTRACT: 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. (AU) | |
| FAPESP's process: | 11/50889-9 - Development and validation of the látex agglutination test (LAT) for diagnosis of canine distemper virus or preparation of RNA for direct use in RT-qPCR |
| Grantee: | João Pessoa Araújo Junior |
| Support Opportunities: | Regular Research Grants |