Partial sequence identification of grapevine-leafroll-associated virus-1 and development of a highly sensitive IC-RT-PCR detection method.
Using immunocapture reverse transcription PCR (IC-RT-PCR) a specific PCR product from GLRaV-1 infected vine samples was amplified with the help of degenerate primers deduced from the conserved HSP70 region of closteroviruses. 511 basepairs of the 5'end of GLRaV-1 HSP70 gene were identified. Within this region, putative GLRaV-1 specific primers were designed and an IC-RT-PCR detection procedure was developed which is about 125 times more sensitive than the established ELISA method. No PCR product was amplified in GLRaV-2,-3 and -4 infected plants which indicates the specificity of the primers. This procedure may serve as an alternative method for GLRaV-1 detection where the sensitivity of ELISA is insufficient.
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Zentrum für Angewandte Genetik, Muthgasse 18, BOKU-Wien, A-1190, Wien, Austria.
Leonhardt
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Steinkellner
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D001483:Base Sequence; D017790:Closterovirus / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers; D018076:DNA, Complementary; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019685:Rosales / Q000821:virology*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D018076:DNA, Complementary; D018840:HSP70 Heat-Shock Proteins