Comparison of ELISA and RT-PCR for the detection of beet yellows closterovirus in plants and aphids

Abstract

A reverse-transcription polymerase chain reaction (RT-PCR) was developed to detect beet yellows closterovirus (BYV) in plants and single aphids using primers spanning the conserved regions of the published sequences of the coat protein gene and open reading frames 7 and 8. Three total RNA extraction procedures were examined and all were found to produce RNA of sufficient quality for RT-PCR, although the RNA extraction kit supplied by Flowgen was found to be the most versatile for the extraction of BYV from individual aphids. When 60 aphids, which had fed on virus infected sugar beet were tested by RT-PCR, 55% of individuals were found to contain BYV. Two groups of 36 individual aphids were tested by TAS-ELISA using a specific BYV monoclonal antibody; 53% gave positive absorbance values when a substrate amplification system was used, but none was found to contain virus when the system was replaced with the substrate p-nitrophenyl phosphate. An immunocapture RT-PCR method was shown to detect BYV regardless of whether the RNA had been extracted from the trapped particles or the reverse transcription and PCR mixtures were added to the wells containing intact particles. The RT-PCR method is now used to determine the numbers of BYV carrying aphids migrating into sugar-beet crops.