hochsensitiver detection using geschachtelter pcr for efficient multiplex detection of members of the vitivirus, foveavirus and closteovirus genus in wine

摘要

The present invention provides for an optimised nested reverse transcription polymerase chain reaction method, that enables rapid and simultaneous detection of members of Vitivirus, Foveavirus and Closterovirus genus in grapevine samples, with high sensitivity, specificity and polyvalence. The method involvcs a "one-step" RT-PCR, in which a combination of degenerate deoxyinosine(dI)-substituted primers amplifies part of the polymerase region of vitiviruses and foveaviruses and part of the HSP 70 homologue gene of closteroviruses, followed by a nested PCR amplification that increases the specificity and sensitivity of detection. The sensitivity of the method was further increased, by using the dI-containing primers along with respective homologous degenerated primers wherein dI is substituted by deoxyguanosine (dG) in regions of sequence homology. This multiplex nested RT-PCR method can reliably co-detect virus species of all three genera in grapevine allowing simple, fast, and cost-effective analysis of a large number of samples. IMAGE