Sequence analysis and detection using immunocapture-PCR of Cymibidium mosaic virus and Odontoglossum ringspot virus in Hawaiian orchids

A Hawaiian isolate of Cymbidium mosaic virus (CyMV-H) was purified from Dendrobium orchid. and a cDNA library was constructed. Clones containing the coat protein (CP) gene and movement protein (MP) gene were identified by colony hybridization and polymerase chain reaction (PCR). The Hawaiian isolate of Odontoglossum ringspot virus (ORSV-H) was purified from Cattleya orchid. The CyMV CP gene was PCR amplified from a cDNA clone. The ORSV CP and 54 kDa putative replicase genes and CyMV-MP gene were cloned by RT-PCR. Sequences of these genes of CyMV-H and ORSV-H were compared with those of CyMV and ORSV from Singapore, Japan, Korea,and Germany. The high degree of sequence identity (91-99%) at the nucleotide level for all gene sequences analysed, shows that CyMV and ORSV from different countries are closely related. Sequence comparison results show that CyMV strains can be divided into two groups based on differences in amino acid sequences of the coat protein gene: CyMV-H closely resembles CyMV-S1 while CyMV-S2 resembles CyMV-K. A sensitive, rapid, and reliable immunocapture PCR (IC-PCR) assay was developed to detect both viruses. CyMV was detected from dilutions equivalent to 100 ng of orchid material and ORSV was detected From dilutions equivalent to 10 mu g of orchid material. IC-PCR was compared with direct binding PCR (DB-PCR) and ELISA for their sensitivities.