Detection and characterization of Plum pox virus: molecular methods

Since their initial description, molecular methods for the detection of Plum pox virus (PPV) have been increasingly used by diagnostic laboratories. Although they initially provided higher sensitivity than traditional serological (ELISA) techniques, molecular hybridization assays were rapidly superseded by RT‐PCR based systems for detecting and characterizing PPV isolates. These techniques have indeed surpassed all traditional methods in sensitivity and specificity. Continuous efforts are being made to improve the use of molecular techniques in routine tests, in particular to overcome specific problems caused by plant material with high contents of RT‐PCR inhibitors. Different systems for viral target preparation before the RT‐PCR reaction have been developed based on immunocapture, or, avoiding extract preparation, on print and squash capture. The use of immobilised targets on paper has allowed the detection of PPV in single aphids. Sequence data from a number of PPV isolates, often obtained through PCR‐based techniques has permitted the clustering of PPV strains into six groups: D, M, EA, C, W and Rec and the development of group‐ or strain‐specific detection assays. Other important advances have arisen with the determination of the viral load in a plant or aphid samples by quantitative, real‐time PCR, as automation and user‐friendly softwares have made this technology more available.