Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP (R) using reverse transcription-recombinase polymerase amplification

摘要

Plum pox virus (PPV) causes the most destructive viral disease known as plum pox or Sharka disease in stone fruit trees. As an important regulated pathogen, detection of PPV is thus of critical importance to quarantine and eradication of the spreading disease. In this study, the innovative development of two AmplifyRP (R) tests is reported for a rapid isothermal detection of PPV using reverse transcription-recombinase polymerase amplification. In an AmplifyRP (R) test, all specific recombination and amplification reactions occur at a constant temperature without thermal cycling and the test results are either recorded in real-time with a portable fluorescence reader or displayed using a lateral flow strip contained inside an amplicon detection chamber. The major improvement of this assay is that the entire test from sample preparation to result can be completed in as little as 20 min and can be performed easily both in laboratories and in the field. The results from this study demonstrated the ability of the AmplifyRP (R) technique to detect all nine PPV strains (An, C, CR, D, EA, M, Rec, T, or W). Among the economic benefits to pathogen surveys is the higher sensitivity of the AmplifyRP (R) to detect PPV when compared to the conventional ELISA and ImmunoStrip assays. This is the first report describing the use of such an innovative technique to detect rapidly plant viruses affecting perennial crops. (C) 2014 Elsevier B.V. All rights reserved.