Direct sample preparation methods for the detection of Plum pox virus by real-time RT-PCR. Validation and practice parameters Authors Nieves Capote Valencian Institute for Agricultural Research (IVIA), Center for Plant Protection and Biotechnology, Valencia Edson Bertolini Valencian Institute for Agricultural Research (IVIA), Center for Plant Protection and Biotechnology, Valencia Antonio Olmos Valencian Institute for Agricultural Research (IVIA), Center for Plant Protection and Biotechnology, Valencia Eduardo Vidal Valencian Institute for Agricultural Research (IVIA), Center for Plant Protection and Biotechnology, Valencia M. Carmen Martínez Valencian Institute for Agricultural Research (IVIA) Mariano Cambra Valencian Institute for Agricultural Research (IVIA) Keywords: Plum pox virus (PPV), sharka disease, spot real-time RT-PCR, winter or dormand diagnostic methods, post-test probability Abstract 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. [Int Microbiol 2009; 12(1):1-6]. Author Biographies Nieves Capote, Valencian Institute for Agricultural Research (IVIA), Center for Plant Protection and Biotechnology, Valencia Valencian Institute for Agricultural Research (IVIA), Center for Plant Protection and Biotechnology, Valencia Edson Bertolini, Valencian Institute for Agricultural Research (IVIA), Center for Plant Protection and Biotechnology, Valencia Valencian Institute for Agricultural Research (IVIA), Center for Plant Protection and Biotechnology, Valencia Antonio Olmos, Valencian Institute for Agricultural Research (IVIA), Center for Plant Protection and Biotechnology, Valencia Valencian Institute for Agricultural Research (IVIA), Center for Plant Protection and Biotechnology, Valencia Eduardo Vidal, Valencian Institute for Agricultural Research (IVIA), Center for Plant Protection and Biotechnology, Valencia Valencian Institute for Agricultural Research (IVIA), Center for Plant Protection and Biotechnology, Valencia M. Carmen Martínez, Valencian Institute for Agricultural Research (IVIA) Valencian Institute for Agricultural Research (IVIA) Mariano Cambra, Valencian Institute for Agricultural Research (IVIA) Valencian Institute for Agricultural Research (IVIA) Downloads PDF Published 2010-09-20 Issue Vol. 12 No. 1 (2009) Section Research Articles License Submission of a manuscript to International Microbiology implies: that the work described has not been published before, including publication in the World Wide Web (except in the form of an Abstract or as part of a published lecture, review, or thesis); that it is not under consideration for publication elsewhere; that all the coauthors have agreed to its publication. 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