Use of reverse transcription-polymerase chain reaction (RT-PCR) for Cymbidium mosaic virus (CyMV) detection in orchids
The reverse transcription-polymerase chain reaction CRT-PCR) was adapted for detection of Cymbidium mosaic virus CCyMV) in orchids. The oligonucleotide primers used were selected from the predicted homologous coat protein region of CyMV and other Potexviruses which enabled to amplify approximate...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2000
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/9129/1/FSAS_2000_33%20IR.pdf |
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| Summary: | The reverse transcription-polymerase chain reaction CRT-PCR) was
adapted for detection of Cymbidium mosaic virus CCyMV) in orchids.
The oligonucleotide primers used were selected from the predicted
homologous coat protein region of CyMV and other Potexviruses
which enabled to amplify approximately 313 bp and 227 bp fragments
using optimum reaction conditions of 2.5 mM MgCh and 30 cycles of
amplification. The RT-PCR allowed the detection of CyMV RNA and virion in
purified fonns as well as in crude tissue extracts of orchid. Direct
CyMV RNA detection was possible in leaves, shoots, stems, roots and
petals. The detection limits of RNA in purified CyMV and virion by
RT-PCR described were 10 ng and 2 ng, respectively. The PCR
amplified fragments were confinned to be CyMV-specific by dotblot
hybridization with DIG-labelled CyMV cDNA probe.
The suitability of the RT-PCR in routine testing of CyMV was
detennined and compared with those of DAS-ELISA. Thirty samples
of leaf tissues representing various genera or hybrids of cultivated
local orchid from glasshouse and commercial nurseries were tested
for CyMV by RT-PCR and DAS-ELISA. Among 15 samples that
tested positive for CyMV infection by DAS-ELISA, only 7 samples
gave the expected amplification fragments when subjected in RTPCR
assays. The equal detection limit on purified CyMV virion by
RT-PCR and DAS-ELISA and lower sensitivity of RT-PCR in
detecting CyMV in a field indexing trial suggested that RT-PCR is
unsuitable to replace DAS-ELISA for routine testing of CyMV in
local orchids. |
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