Canine distemper virus detection by different methods of One-Step RT-qPCR
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.
Main Authors: | , , , |
---|---|
Format: | Digital revista |
Language: | English |
Published: |
Universidade Federal de Santa Maria
2016
|
Online Access: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0103-84782016000901601 |
Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
id |
oai:scielo:S0103-84782016000901601 |
---|---|
record_format |
ojs |
spelling |
oai:scielo:S0103-847820160009016012016-08-12Canine distemper virus detection by different methods of One-Step RT-qPCRTozato,Claudia de CamargoZadra,Vívian FerreiraBasso,Caroline RodriguesAraújo Junior,João Pessoa canine distemper molecular diagnosis One-Step RT-qPCR urine centrifugation 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.info:eu-repo/semantics/openAccessUniversidade Federal de Santa MariaCiência Rural v.46 n.9 20162016-09-01info:eu-repo/semantics/articletext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0103-84782016000901601en10.1590/0103-8478cr20150932 |
institution |
SCIELO |
collection |
OJS |
country |
Brasil |
countrycode |
BR |
component |
Revista |
access |
En linea |
databasecode |
rev-scielo-br |
tag |
revista |
region |
America del Sur |
libraryname |
SciELO |
language |
English |
format |
Digital |
author |
Tozato,Claudia de Camargo Zadra,Vívian Ferreira Basso,Caroline Rodrigues Araújo Junior,João Pessoa |
spellingShingle |
Tozato,Claudia de Camargo Zadra,Vívian Ferreira Basso,Caroline Rodrigues Araújo Junior,João Pessoa Canine distemper virus detection by different methods of One-Step RT-qPCR |
author_facet |
Tozato,Claudia de Camargo Zadra,Vívian Ferreira Basso,Caroline Rodrigues Araújo Junior,João Pessoa |
author_sort |
Tozato,Claudia de Camargo |
title |
Canine distemper virus detection by different methods of One-Step RT-qPCR |
title_short |
Canine distemper virus detection by different methods of One-Step RT-qPCR |
title_full |
Canine distemper virus detection by different methods of One-Step RT-qPCR |
title_fullStr |
Canine distemper virus detection by different methods of One-Step RT-qPCR |
title_full_unstemmed |
Canine distemper virus detection by different methods of One-Step RT-qPCR |
title_sort |
canine distemper virus detection by different methods of one-step rt-qpcr |
description |
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. |
publisher |
Universidade Federal de Santa Maria |
publishDate |
2016 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0103-84782016000901601 |
work_keys_str_mv |
AT tozatoclaudiadecamargo caninedistempervirusdetectionbydifferentmethodsofonesteprtqpcr AT zadravivianferreira caninedistempervirusdetectionbydifferentmethodsofonesteprtqpcr AT bassocarolinerodrigues caninedistempervirusdetectionbydifferentmethodsofonesteprtqpcr AT araujojuniorjoaopessoa caninedistempervirusdetectionbydifferentmethodsofonesteprtqpcr |
_version_ |
1756406415140847616 |