Articles
PROPOSAL FOR A NEW INTERNATIONAL PROTOCOL FOR DETECTION AND IDENTIFICATION OF PLUM POX VIRUS: VALIDATION OF THE TECHNIQUES
Article number
781_27
Pages
181 – 192
Language
English
Abstract
An update of the European and Mediterranean Plant Protection Organization (EPPO) protocol for the specific diagnostic of Plum pox virus (PPV) has been elaborated by a nominee working group from different organizations.
The draft protocol was elaborated for the International Plant Protection Convention (IPPC) governed by the Interim Commission on Phytosanitary Measures hosted by FAO. The protocol includes biological indexing and serological and molecular assays as screening tests.
The selected protocols for detection of any PPV isolate are: 1) Biological indexing: inoculation of woody indicators plants (GF305 or Nemaguard peach seedlings or Prunus tomentosa); 2) Serological tests: DASI-ELISA with 5B-IVIA universal monoclonal antibody or DAS-ELISA with 5B-IVIA or polyclonal antibodies; and 3) Molecular tests: IC-RT-PCR (using P1/P2 or 3NCR primers) or Co-PCR (using P10/P20/P1/P2 primers and hybridization with universal probes) or real-time RT-PCR (using SYBR Green and/or TaqMan chemistries). The combination of two different screening methods (with the validated and recommended protocols and reagents), is required to officially support a positive PPV detection.
Identification tests are also included in the protocol for typing PPV isolates into D, M, EA, C, W and Rec groups.
For first or critical findings of PPV, coat protein gene (and NIb gene in the case of PPV-Rec) sequences should be reported.
The protocol and some techniques and reagents were validated in ring-tests.
The draft protocol was elaborated for the International Plant Protection Convention (IPPC) governed by the Interim Commission on Phytosanitary Measures hosted by FAO. The protocol includes biological indexing and serological and molecular assays as screening tests.
The selected protocols for detection of any PPV isolate are: 1) Biological indexing: inoculation of woody indicators plants (GF305 or Nemaguard peach seedlings or Prunus tomentosa); 2) Serological tests: DASI-ELISA with 5B-IVIA universal monoclonal antibody or DAS-ELISA with 5B-IVIA or polyclonal antibodies; and 3) Molecular tests: IC-RT-PCR (using P1/P2 or 3NCR primers) or Co-PCR (using P10/P20/P1/P2 primers and hybridization with universal probes) or real-time RT-PCR (using SYBR Green and/or TaqMan chemistries). The combination of two different screening methods (with the validated and recommended protocols and reagents), is required to officially support a positive PPV detection.
Identification tests are also included in the protocol for typing PPV isolates into D, M, EA, C, W and Rec groups.
For first or critical findings of PPV, coat protein gene (and NIb gene in the case of PPV-Rec) sequences should be reported.
The protocol and some techniques and reagents were validated in ring-tests.
Publication
Authors
M. Cambra, N. Capote, A. Olmos, E. Bertolini, M.T. Gorris, N.L. Africander, L. Levy, S.L. Lenardon, G. Clover, D. Wright
Keywords
PPV types, serological methods, molecular methods, OEPP protocol, ring-test, real-time PCR
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