Rapid detection of cymbidium mosaic virus by the polymerase chain reaction (PCR)

Abstract

A direct, simple and sensitive method was developed for the detection of cymbidium mosaic virus (CyMV), based on the polymerase chain reaction (PCR). Two oligonucleotide primers were selected from regions that are homologous to potexviruses and CyMV, and used to hybridize with purified viral RNA and particles. This resulted in the amplification of a 313 bp fragment after 30 cycles of PCR in all samples. A less prominent fragment of 227 bp was also obtained due to mispriming of the second primer. The amplified fragments were easily seen in an agarose gel when stained with ethidium bromide. As little as 1 fg of viral RNA (about 200 target copies) or 10 fg of purified virus (approximately 130 viral particles) were detectable. For CyMV infected orchid leaf tissues, 10 μl aliquots of 1 mm3 tissue homogenate in 1 ml could be detected routinely. All reverse transcription and amplification reactions were carried out in a single tube and results can be obtained within 5 h. © 1993.