Canine distemper virus detection by different methods of One-Step RT-qPCR

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:...

Full description

Saved in:
Bibliographic Details
Published in:Ciência rural 2016-09, Vol.46 (9), p.1601-1606
Main Authors: Tozato, Claudia de Camargo, Zadra, Vívian Ferreira, Basso, Caroline Rodrigues, Araújo Junior, João Pessoa
Format: Article
Language:English
Subjects:
AGRONOMY
Annealing
Binding sites
Canine distemper
Centrifugation
centrifugação
cinomose canina
Diagnosis
diagnóstico molecular
Dilution
Laboratories
Mathematical analysis
One-Step RT-qPCR
Polymerase chain reaction
Research projects
Ribonucleic acid
RNA
RNA viruses
Sensitivity analysis
Test animals
Ultracentrifugation
urina
Urine
Viruses
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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. RESUMO: Três kits comerciais de One-Step RT-qPCR foram avaliados para o diagnóstico molecular do Vírus da Cinomose Canina.Utilizando o kit que apresentou melhor desempenho, dois sistemas de RT-PCR em tempo real (RT-qPCR) foram comparados quanto à sensibilidade analítica na detecção do RNA do Vírus da Cinomose Canina:One-Step RT-qPCR (Sistema A) e One-Step RT-qPCR seguido da NESTED-qPCR (Sistema B).Os limites de detecção dos dois sistemas foram determinados utilizando diluição seriada de RNA sintético do Vírus da Cinomose Canina ou de uma amostra de urina positiva. Adicionalmente, uma amostra de urina foi avaliada com centrifugação ou ultracentrifugação prévia. Os kits comerciais de One-Step RT-qPCR amplificaram o RNA do vírus da cinomose canina em 10 (100%) amostras de urinas de animais sintomáticos. O kit de One-Step RT-qPCR que apresentou melhor resultado foi utilizado para aval
ISSN:1678-4596
0103-8478
1678-4596
DOI:10.1590/0103-8478cr20150932