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We established a simple and reliable RT-PCR method for the detection of orchids viruses. This method was used to determine cymbidium mosaic virus (CyMV) and odontoglossum ringspot virus (ORSV) in Cybidium hoosai Makino. Primers were designed for PCR products: 327 bp, 33l bp in coat protein gene of CyMV and ORSV RT-PCR reaction mixture was one step mixture, containing 20 pmol of primer, 30 units of reverse transcriptase, 5 unit of RNasin, and the crude RNA extracts. During RT-PCR, reaction mixture was incubated at 95℃ for 5 minutes followed by quick incubation in ice bath and subsequently incubated at 42℃ for 30 min for cDNA synthesis. The PCR reaction was carried out in 1 cycle at 96℃ for 3 minutes, 60℃ for 1 minute, 72℃ 2 minutes, followed by 40 cycles at 96℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 1 minute, and finally at 72℃ for 10 minutes. By using the CyMV, ORSV primers produced, specific PCR products were confirmed in virus infected orchids, while no specific products were detected in virus free orchids. Nucleotide sequence of coat protein genes of CyMV and ORSV shared 94-98% and 97-99% respectively. RT-PCR method is a more efficient assay, and it was 100 times more sensitive than ELISA. The RT-PCR method established in this experiment should lead to a quick diagnose of CyMV or ORSV.
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