Real-time RT-PCR (TaqMan) assays for the detection of viruses associated with Rugose wood complex of grapevine.
J Virol Methods
; 154(1-2): 69-75, 2008 Dec.
Article
en En
| MEDLINE
| ID: mdl-18848580
ABSTRACT
Real-time TaqMan RT-PCR (TaqMan RT-PCR) assays were developed to detect the viruses associated with Rugose wood complex of grapevines. The viruses detected were Rupestris stem pitting-associated virus in the genus Foveavirus, Grapevine virus A, Grapevine virus B and Grapevine virus D in the genus Vitivirus. The coat protein was found to be the most conserved gene within the viral species, therefore, the primers and probes for TaqMan RT-PCR assays were designed from the multiple alignment of the coat protein sequence of various isolates of each virus. Comparisons were also made between the conventional one step RT-PCR and TaqMan RT-PCR for the detection of these viruses using four-fold serial dilutions of both purified RNA and crude extract prepared from grapevine tissue. Results showed that TaqMan RT-PCR was more sensitive and could detect viruses at 32- and 256-fold higher dilutions for purified RNA and crude extract, respectively, compared to RT-PCR. The use of an internal control (18S rRNA) allowed comparison of sample preparation protocols and amplification efficiencies between samples.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Enfermedades de las Plantas
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Virosis
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Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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Vitis
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Flexiviridae
Tipo de estudio:
Diagnostic_studies
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Evaluation_studies
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Risk_factors_studies
Idioma:
En
Año:
2008
Tipo del documento:
Article