Quantitation of Grapevine leafroll associated virus-1 and -3, Grapevine virus A, Grapevine fanleaf virus and Grapevine fleck virus in field-collected Vitis vinifera L. 'Nebbiolo' by real-time reverse transcription-PCR.

TaqMan one-step real-time qRT-PCR assays were developed for the quantitation of Grapevine leafroll associated virus-1 and -3 (GLRaV-1 and -3), Grapevine virus A (GVA), Grapevine fanleaf virus (GFLV) and Grapevine fleck virus (GFkV) in Vitis vinifera L. Virus load in the progenies of three 'Nebbiolo' clones planted in two experimental vineyards in Piemonte (northwestern Italy), and carrying the viruses in different combinations, was evaluated. Quantitation primers were designed on the RNA-dependent RNA polymerases (RdRp) of each virus to exclude the amplification of subgenomic mRNAs. Viral quantity was referred to as the concentration of the V. vinifera glyceraldehyde-3P-dehydrogenase (GAPDH) housekeeping gene. A TaqMan protocol for the quantitation of the 'Nebbiolo' GAPDH mRNA was also optimised. The absolute quantitation of viral RNA and GAPDH mRNA was achieved using external standard curves from 10-fold dilutions of viral RdRp in vitro transcripts, ranging between 10(9) and 10(3) RNA copies. The relative quantity of viral genome units per GAPDH mRNA copy was calculated as the difference between the Log virus quantity and the corresponding Log GAPDH transcript quantity. The mean load of each virus was determined for 10 infected vines and ranged between 3 (GLRaV-1 and GFLV) and 5700 (GFkV) viral genomes per 100 V. vinifera GAPDH transcripts, with GLRaV-3 and GVA within this range.