Comparison of different methods of RNA isolation for plum pox virus detection by reverse transcription polymerase chain reaction

The diagnosis of plum pox virus (PPV) is still considered one of the most i mportant aspects of the "sharka" problem. In fact, different studies demons trated an uneven distribution of the virus in infected trees due to a high variability in virus concentration. These aspects complicate the PPV diagno sis. To date, biological, serological and molecular assays have been succes sively developed in order to obtain sensitive and efficient PPV detection t echniques. In particular, the polymerase chain reaction (PCR) technique see ms to be promising and can be considered the most sensitive and reliable on e. Preparation of viral RNA is still a fundamental step in reverse transcri ption-PCR (RT-PCR) technique, especially when applied to large scale testin g, i.e., for certification purposes. In order to find the most rapid and ef ficient procedure, we have compared three different procedures of extractio n of viral RNA to be processed RT-PCR. Their common characteristics is thei r capacity to extract the RNA from a small amount of plant tissue without o rganic solvents in the extraction fluid. The procedures were as follows: an immune-capture (IC) method using a specific antiserum, a silica-capture (S C) method using a non-specific matrix, and a simple and rapid RNA extractio n (RE) method. They all were followed by one-tube RT-PCR. The obtained resu lts show that all the three techniques allowed a successful amplification a nd detection of PPV in tested samples except the SC-PCR method which proved less effective. In fact, the IC-PCR and RE-PCR methods amplified and detec ted PPV in all isolates tested, while the SC-PCR method was able to reveal the presence of the virus in apricot and infected control samples only.