Journal of Taiwan Agricultural Research

Author:Chin-Chih Chen* and Fen-Lang Chiang

Abstract:

    Cymbidium ringspot virus (CymRSV) is recorded in foreign countries, but not found in Taiwan. The molecular and serological detection reagents for the inspection of CymRSV on orchids were developed in this study. In molecular detection, two primer pairs for reverse transcription-polymerase chain reaction (RT-PCR) were designed based on the nucleotide sequences of CymRSV coat protein (CP). The primer pair CyRcpu/CyRcpd was designed for amplifying the full-length CP gene and its flanking borders. The primer pair CyRu/CyRd was used to amplify the conserved region of the CymRSV-CP gene. In RT-PCR, the expected 1370-bp and 540-bp DNA fragments were amplified by the primer pairs CyRcpu/CyRcpd and CyRu/CyRd, respectively, from the commercial DSMZ-CymRSV RNA. The annealing temperature under the 60℃, the primer pair CyRu/CyRd was used to specifically detect CymRSV in RT-PCR. A set of four primers (CyR-FIP/CyR-BIP/CyR-F3/CyR-B3) was successfully used to amplify CymRSV RNA under 65℃ for 1 h in reverse transcription loop-mediated isothermal amplification (RT-LAMP), and the results were able to be determined by naked eye observation. RT-LAMP with a specificity for CymRSV detection and has a 25-time higher sensitivity than RT-PCR assay. In serological detection, the predicted 41.8 kDa of the bacterial expressed fusion protein (BEP189) was positively reacted to two commercial CymRSV antibodies. The CymRSV antiserum (#189) was prepared from the rabbit immunized with the bacterial-expressed CymRSV-CP. In the detections of CymRSV by the ways of indirect enzyme-linked immunosorbent assay (indirect ELISA) and western blotting, the prepared antibody #189 specifically reacted with its expressed CP as well as with the commercial controls of CymRSV, but not reacted to the healthy orchid tissues and other 9 viruses on orchids. This is the first report to develop the RT-LAMP method for detecting CymRSV and to apply the RT-PCR assay for inspecting CymRSV on orchids. Moreover, the prepared polyclonal antibody against CymRSV is efficient to differentiate CymRSV from other 9 tested orchid viruses.

Key words: Cymbidium ringspot virus (CymRSV), Serological detection, RT-PCR detection, RTLAMP

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