Detecção do vírus da cinomose canina por diferentes métodos de one-step RT-qPCR

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dc.contributor.authorTozato, Claudia de Camargo [UNESP]
dc.contributor.authorZadra, Vívian Ferreira [UNESP]
dc.contributor.authorBasso, Caroline Rodrigues [UNESP]
dc.contributor.authorAraújo Junior, João Pessoa [UNESP]
dc.contributor.institutionUniversidade Estadual Paulista (Unesp)
dc.date.accessioned2018-12-11T17:04:02Z
dc.date.available2018-12-11T17:04:02Z
dc.date.issued2016-09-01
dc.description.abstractThree 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.en
dc.description.affiliationDepartamento de Microbiologia e Imunologia Instituto de Biociências Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)
dc.description.affiliationDepartamento de Química e Bioquímica Instituto de Biociências Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)
dc.description.affiliationInstituto de Biotecnologia (IBTEC) Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)
dc.description.affiliationUnespDepartamento de Microbiologia e Imunologia Instituto de Biociências Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)
dc.description.affiliationUnespDepartamento de Química e Bioquímica Instituto de Biociências Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)
dc.description.affiliationUnespInstituto de Biotecnologia (IBTEC) Universidade Estadual Paulista Júlio de Mesquita Filho (UNESP)
dc.format.extent1601-1606
dc.identifierhttp://dx.doi.org/10.1590/0103-8478cr20150932
dc.identifier.citationCiencia Rural, v. 46, n. 9, p. 1601-1606, 2016.
dc.identifier.doi10.1590/0103-8478cr20150932
dc.identifier.fileS0103-84782016000901601.pdf
dc.identifier.issn1678-4596
dc.identifier.issn0103-8478
dc.identifier.scieloS0103-84782016000901601
dc.identifier.scopus2-s2.0-84977584474
dc.identifier.urihttp://hdl.handle.net/11449/173188
dc.language.isoeng
dc.relation.ispartofCiencia Rural
dc.relation.ispartofsjr0,337
dc.rights.accessRightsAcesso aberto
dc.sourceScopus
dc.subjectCanine distemper
dc.subjectCentrifugation
dc.subjectMolecular diagnosis
dc.subjectOne-Step RT-qPCR
dc.subjectUrine
dc.titleDetecção do vírus da cinomose canina por diferentes métodos de one-step RT-qPCRpt
dc.title.alternativeCanine distemper virus detection by different methods of one-step RT-qPCRen
dc.typeArtigo

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