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.