[show abstract][hide abstract] ABSTRACT: Antisera against important orchid viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV), were separately produced using bacterially expressed recombinant capsid proteins (CP), instead of purified virus
particles, as immunogens. These antisera were then designated as home-made CymMV CP antiserum (HM-Cy) and home-made ORSV CP
antiserum (HM-OR). The high specificity of HM-Cy and HM-OR were confirmed by immunoblot. Their detection limits were determined
using indirect-enzyme-linked immunosorbent assay (I-ELISA). Both HM-Cy and HM-OR showed low background reactivity to healthy
plants and thus displayed a high S/H ratio (sample OD405/healthy control OD405) in tested orchids. The data indicated that
our antisera were efficient and accurate in determination of negative and positive results in ELISA test as commercial antibodies.
Therefore, these home-made antisera of CymMV and ORSV are suitable for the certification programme of orchids due to their
low cost and high specificity. HM-Cy and HM-OR were further used for a field survey to study the incidence of CymMV and ORSV.
The results showed that CymMV is more prevalent than ORSV in Taiwan.
European Journal of Plant Pathology 04/2012; 122(2):297-306. · 1.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dasheen mosaic virus (DsMV), Turnip mosaic virus (TuMV), Konjac mosaic virus (KoMV) and Zantedeschia mild mosaic virus (ZaMMV) are important potyviruses previously identified in calla lily plants in Taiwan. In order to save time and cost of
virus detection, a multiplex RT-PCR assay was developed for these calla potyviruses. Specific primers for each virus were
designed based on the sequences of 3′ terminal region of respective viruses. To prevent false negative results, a primer pair
specific to plant mitochondrial nad5 mRNA was used to produce a 185-bp fragment as an internal control of RT-PCR. The specificities of primers were confirmed
by means of simplex and multiplex PCR assays. Optimal primer concentration ratio was identified by multiplex PCR assay. Total
RNAs purified from virus-infected plants were used directly or mixed in different combinations, and then tested by multiplex
RT-PCR. The result indicated that the expected RT-PCR products could be specifically amplified and identified on the basis
of their molecular sizes. The detection sensitivity of multiplex RT-PCR was 25–625 times higher than that of indirect-ELISA
(I-ELISA) depending on the virus. When applied to field surveys, multiplex RT-PCR could detect more single as well as mixed
infection samples than I-ELISA. Accordingly, our multiplex RT-PCR assay provides a simple, rapid and reliable method for multiple
potyvirus detection in calla lily.
European Journal of Plant Pathology 04/2012; 126(1):43-52. · 1.61 Impact Factor