A spot nested RT-PCR method for the simultaneous detection of members of the Vitivirus and Foveavirus genera in grapevine

A reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to allow rapid, and simultaneous detection of Vitivirus and Foveavirus sequences in two steps. The method involved a one-step RT-PCR, in which the combination of degenerate deoxyinosine-substituted primers amplified par...

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Bibliographic Details
Published in:Journal of virological methods 2003, Vol.107 (1), p.99-106
Main Authors: Dovas, C.I, Katis, N.I
Format: Article
Language:English
Subjects:
Biological and medical sciences
Degenerate primers
Deoxyinosine
Diagnosis
Foveavirus
Fundamental and applied biological sciences. Psychology
Generalities. Techniques. Transmission, epidemiology, ecology. Antiviral substances, control
Grapevine virus
Phytopathology. Animal pests. Plant and forest protection
Plant Viruses - isolation & purification
Plant viruses and viroids
Reverse Transcriptase Polymerase Chain Reaction - methods
Sensitivity and Specificity
Spot Nested RT-PCR
Templates, Genetic
Vitis - virology
Vitivirus
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Summary:A reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to allow rapid, and simultaneous detection of Vitivirus and Foveavirus sequences in two steps. The method involved a one-step RT-PCR, in which the combination of degenerate deoxyinosine-substituted primers amplified part of the polymerase region of both genera, followed by a nested PCR amplification that increased specificity and sensitivity of detection. The increase in sensitivity also permitted the use of a simple and rapid template preparation protocol, involving the spotting of plant sap extract on a nylon membrane. Consistent amplification with infected grapevine plants was possible after inclusion of additives for inhibiting polyphenolic compounds during template preparation. This spot nested RT-PCR method can reliably detect virus species of both genera in grapevine allowing simple, fast, and cost-effective analysis of a large number of samples in certification schemes.
ISSN:0166-0934
1879-0984
DOI:10.1016/S0166-0934(02)00197-0