Rapid detection and identification of odontoglossum ringspot virus by polymerase chain reaction amplification

The odontoglossum ringspot Tobamovirus (ORSV) movement and coat proteins genes were selected for the design of oligonucleotide primers for amplification of a 1,085 bp fragment. A combined assay of reverse transcription and the polymerase chain reaction (RT-PCR) was performed with 20-mer ORSV-specific primers and crude nucleic acid extracts from virus-infected orchids for rapid detection of the virus. The lowest concentration of template viral RNA required for detection was 10 fg. The RT-PCR is a 10(3) times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. No PCR product was observed when cymbidium mosaic potexvirus or a crude extract of healthy Cymbidium sp. were used as a template in RT-PCR with the same primers. The specificity of the primers was verified using other tobamoviruses RNAs.