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根据已报道的建兰花叶病毒(CyMV)和齿兰环斑病毒(ORSV)基因组核苷酸序列,在其cp基因上下游设计PCR引物。CyMV预计扩增产物784bp,ORSV预计扩增产物604bp。以采集自广东省顺德的墨兰和文心兰表现病毒病症状的病株叶组织总RNA为模板,进行RT-PCR扩增。对预期大小的5个扩增产物进行克隆和测序,结果表明,来源于不同兰种或同一兰种不同兰场的病样CyMV引物扩增产物核苷酸序列存在少量差异,但均与世界各地的CyMV分离物cp基因高度同源;而来源于不同兰种的病样ORSV引物扩增产物核苷酸序列完全相同,与世界各地的ORSV分离物cp基因高度同源。因此可将侵染广东兰花的两种病毒鉴定为CyMV和ORSV。混合上述两种病毒的 PCR引物,采用双重RT-PCR扩增,对采自广东顺德23个兰场共153份样品进行病毒检测,76份(49.7%)检出CyMV,52份(34.0%)检出ORSV,2份(1.3%)同时检出CyMV和ORSV。
PCR primers were designed upstream and downstream of cp gene according to reported nucleotide sequences of Cymbidium glaucoma (CyMV) and torenia ring spot virus (ORSV). CyMV expected amplification products 784bp, ORSV expected amplification products 604bp. The total RNA of the leaves of diseased plants collected from Cymbidium and Oncidium in Shunde, Guangdong Province, was used as a template for RT-PCR amplification. The cloning and sequencing of the five amplification products of the expected size showed that the nucleotide sequences of the amplified products of the disease-like CyMV primers derived from different orchid species or the same orchid species had a slight difference, Of CyMV isolates cp gene highly homologous; and ORSV primers derived from different species of ORSV primers nucleotide sequence is identical, and the ORSV isolates cp gene is highly homologous around the world. Therefore, two viruses that infect Guangdong orchids can be identified as CyMV and ORSV. A total of 153 samples collected from 23 orchards in Shunde of Guangdong were detected by double-stranded PCR (RT-PCR). CyMV was detected in 76 (49.7%), 52 (34.0% ORSV was detected and 2 copies (1.3%) of CyMV and ORSV were detected simultaneously.