Optimization of Nucleic Acid Extraction and RT-PCR for Detection of Grapevine fanleaf virus Isolates from Vineyards in North-West of Iran

One of the inhibitors of polymerase chain reaction (PCR) in detecting fruit tree viruses including grapevine viruses is the presence of phenolic compounds and polysaccarides in the plant tissue. These inhibitors degrade the extracted nucleic acid and inactivate polymerases during PCR reaction. In this study, two protocols were examined to extract total RNA from the grapevines suspected to be infected by Grapevine fanleaf virus (GFLV). One protocol was based on phenol chloroform extraction but the other did not use such solvents. In optimization of the RT-PCR reaction, synthesis of first strand cDNA was initially performed by either a virus-specific primer (S2515 or 3300) or oligo d(T)16 and subsequently PCR was undertaken with two primer sets CP433V/ CP91 C or S2515/A3300. RT-PCR on phenol-chloroform extracted RNA did not result in any amplification from grapevine samples although the expected DNA fragments were amplified from GFLVinfected Chenopodium quinoa plants. Presumably, the phenol-based protocol was not able to remove the preventive substances from the extraction and they inhibited the RT-PCR reaction. However, RT with oligo d(T)16 on the RNA which was prepared without using phenol or chloroform from the grapevines and subsequent PCR with CP433V/ CP912C or S2515/ 3300 resulted in amplification of the expected 480- or 810- bp DNA fragment, respectively. This study, for the first time, revealed the extent of GFLV distribution in vineyards of East Azarbaijan and Ardabil provinces in north-west of Iran plus providing ground for molecular characterization of the virus isolates.