Direct sample preparation methods for the detection of Plum pox virus by real-time RT-PCR.
Direct systems to process plant materials allowed high-throughput testing of Plum pox virus (PPV) by real-time reverse transcription (RT)-PCR without nucleic acids purification. Crude plant extracts were diluted in buffer or spotted on membranes to be used as templates. Alternatively, immobilized PPV targets were amplified from fresh sections of plant tissues printed or squashed onto the same supports, without extract preparation. Spot real-time RT-PCR was validated as a PPV diagnostic method in samples collected during the dormancy period and showed high sensitivity (93.6%), specificity (98.0%), and post-test probability (97.9%) towards sharka disease. In an analysis of 2919 Prunus samples by spot real-time RT-PCR and DASI-ELISA 90.8% of the results coincided, demonstrating high agreement (k = 0.77 +/- 0.01) between the two techniques. These results validate the use of immobilized PPV targets and spot real-time RT-PCR as screening method for largescale analyses.
Valencian Institute for Agricultural Research, Center for Plant Protection and Biotechnology, Valencia, Spain.
Nieves
N
Capote
Edson
E
Bertolini
Antonio
A
Olmos
Eduardo
E
Vidal
Maria Carmen
MC
Martínez
Mariano
M
Cambra
D053000:Analytic Sample Preparation Methods; D004797:Enzyme-Linked Immunosorbent Assay; D010935:Plant Diseases / Q000821:virology*; D010944:Plants / Q000821:virology; D017801:Plum Pox Virus / Q000302:isolation & purification*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity