Serological techniques in indexing orchid
2003
Barcial, M.P. | Bajet, N.B. (Philippines Univ. Los Banos, College, Laguna (Philippines))
The serological and biochemical properties of CymMV (Cymbidium mosaic potexvirus) and ORSV (odontoglossum ringspot virus) in the project "Application of serological techniques in indexing orchid plants for viruses" from 1996 to 1999 was successfully purified and characterized. Six trainings on the serological detection of orchid viruses were conducted in 1999 by the project staff. A total of 73 participants, staff and/or owners of orchid nurseries and staff of government agencies (i.e., DA [Department of Agriculture, Quezon City, Philippines]) attended the training. A hands-on training on ELISA (enzyme-linked immunosorbent assay) technique was conducted to develop skills in applying this assay. Visits to nurseries were part of the training so that participants would be able to recognize the typical symptoms of virus infection and learn how to properly collect samples for detection. CymMV was isolated from field-infected Mokara "Saleha", which tested positive for CymMV in ELISA protocol. SDS-PAGE analysis showed the putative CymMV coat protein as the major protein band with a molecular weight of 27.5 kDA based on the relative mobility in 12.5% polycarylamide gel. The identity of this band was confirmed when it gave a positive signal to CymMV antiserum in the western blot analysis. ORSV was purified from field-infected Oncidium "gower Ramsey", which were doubly infected with CymMV. Serological assay of the partially purified virus preparation was a mixture of CymMV and ORSV. The SDS-PAGE analysis showed the putative ORSV coat protein as the major protein band with a molecular weight of 17.3 kDA based on the relative mobility in 12.5% polyacrylamide gel. The identity of this band was confirmed since there was a positive signal to ORSV antiserum in western blot analysis. The locally produced antisera of CymMV and ORSV were comparable to the antisera from Hawaii and Japan, respectively. The use of antisera enabled the researchers to differentiate the healthy from the infected samples
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