| Summary: | 碩士 === 朝陽科技大學 === 生物技術研究所 === 96 === Tissue culture plantlets have become the major exportation item for Taiwan’s Phalaenopsis industry in recent years. In order to maintain the competitiveness of this emerging business continuously, it is very important to ensure the quality and healthiness of the Phalaenopsis plantlets produced in Taiwan. At present, Odontoglossum ringspot virus (ORSV) and Cymbidium mosaic virus (CymMV) are recognized as the most widely spread and economically important viral agents to orchid industry among the 30 known viruses infecting orchids. Nowadays, enzyme-linked immunosorbent assay (ELISA) is the most commonly used virus detection technique in orchid industry worldwide. Traditionally, at least two antisera each specific to ORSV and CymMV are required to perform routine virus detection in ELISA. Traditionally, if there was an antiserum that can detect ORSV and CymMV simultaneously, the cost of time and materials will be reduced into at least half to conduct a regular ELISA test. In a previous study, our laboratory has successfully constructed four artificial recombinant proteins, namely CyOrN, OrCyN, CyOrH, and OrCyH, each all containing partial coat protein sequences of both ORSV and CymMV. Recombinant proteins CyOrN and OrCyN were constructed by linking N-termianl sequences of CP of either CymMV or ORSV with C-terminal sequences of CP of the other virus. On the other hand, proteins of CyOrH and OrCyH were constructed by fusing N-terminal sequences of CP of either CymMV or ORSV with the N-terminal sequences of other virus. These four recombinant proteins were shown to possess both antigenicity of coat proteins of ORSV and CymMV, and consequently the homologous, antisera of these four recombinant proteins could react with both ORSV and CymMV in ELISA, SDS-immunodiffusion and Western blotting tests. In this study, we attempted to prepare the other two possibilities of recombinant proteins (CyOrC and OrCyC) with combination of C-terminal sequences of ORSV and CymMV. Our result comfirmed that the bacterial expressed recombinant proteins of CyOrC and OrCyC also possessed both antigenicities of ORSV and CymMV, and the antisera prepared against these two new recombinant proteins could also detect both viruses in ELISA, SDS-immunodiffusion and Western blotting tests. To test the sensitivity and specificity of the antisera against six different recombinant proteins and compare with two other mono-specific antisera against ORSV and CymMV, we conducted a series indirect ELISA tests on a total of 160 orchid plants in our collection. For those non-detected samples by ELISA, we performed RT-PCR tests to confirm their infection status. The experiment was repeated three times at March 31, April 3, 2007, and January 8, 2008, respectively. Final results showed antiserum against CyOrN recombinant protein had highest sensitivity of 99% and specificity of 100 %. The other five antisera against five other recombinant protein constructions also had fairly high sensitivities (97-98%), except the lowest (94%) one of CyOrH. Furthermore, in these three experiments all six antisera consistently showed higher specificities (98-100%) than the traditionally made antisera against purified virus particles (97% in average). Putting together all these results, the antisera prepared against recombinant proteins containing partial sequences from coat protein gene of ORSV and CymMV could be practically applied in indirect ELISA for simultaneous detection of ORSV and CymMV. The results also implicated that recombinant proteins containing N-terminal sequence of CymMV down streamed with C-terminal sequence of ORSV had highest sensitivity and specificity which is comparable to those of mono-specific traditionally made antisera.
In the second part of this study, we tested the feasibility of using the six antisera specific to both ORSV and CymMV in immuno-captured RT-PCR (IC-RT-PCR) for capturing particles of both viruses. In the experiment, equal volume mixture of the two traditionally made antisera mono-specific to ORSV and CymMV were used for comparison. The result showed using the mixture of two mono-specific antisera in IC-RT-PCR, the sensitivity of 1 pg-100 fg/ml of virus detection could be reached. However, with the double specificity antisera to either of the six recombinant proteins, the sensitivity of detection was about 100 times lower than the mixture of two mono-specific antisera. This is probably due to the differences in the trueness of tertiary structure of epitopes residing in the artificially constructed recombinant proteins comparing with those in the intact virus particles. Nevertheless, an feasible and efficient IC-RT-PCR protocol for simultaneous detection of ORSV and CymMV was developed. In the developed protocol, we improved a procedure of 65 C dry heat treatment for 10 min that could release effectively the viral RNA from captured virions. We also found that by applying RNase inhibitor in the reaction mixture, consistent amplification result of RT-PCR could be improved. In addition, we found that the antibody coated eppendorf tubes could be preserved for at least 6 months without any detrimental effect on the immuno-capturing and later the RT-PCR amplification. Therefore, by the use of antibody pre-coated tubes, IC-RT-PCR could be performed immediately after sample arrival, saving the tedious RNA purification procedures. Furthermore, the developed IC-RT-PCR procedure could also be used for the detection of at least three other viruses, i.e. Cucumber mosaic virus, Jasmine virus T and Hippeastrum mosaic virus. Although the developed IC-RT-PCR procedure seemed to be feasible for simultaneous detection of ORSV and CymMV in orchid tissues, we did encounter some orchid samples that could not be detected by our IC-RT-PCR protocol. Apparently, these samples accumulated very low concentration of ORSV or CymMV virions in their tissues that beyond the sensitivity level of our IC-RT-PCR. Secondly, these samples might be at the begining stage of infection that contained only RNAs in their tissues so that they could not be detected by IC-RT-PCR. This findings suggested that at certain crucial stage such as virus indexing of orchid mother stocks before mericloning, conventional RT-PCR should be a better choice than IC-RT-PCR. Nevertheless, with the improvement accomplished in this study, the developed IC-RT-PCR is certainly a good alternative as virus indexing techniques especially suitable for large number of sampling during field survey.
|
|---|
