ISHS Acta Horticulturae 472: XVII International Symposium Virus and Virus-Like Diseases of Temperate Fruit Crops
EVALUATING THE USE OF IMMUNOCAPTURE AND SAP-DILUTION PCR FOR THE DETECTION OF PRUNUS NECROTIC RINGSPOT VIRUS |
| Authors: | A. Rosner, Y. Shiboleth, S. Spiegel, L. Krisbai, M. Kölber |
| Keywords: | PNRSV, peach, ilarvirus, PCR |
| Abstract:
Reaction conditions were investigated for the detection of PNRSV in peach trees using immunocapture-polymerase chain reaction (IC-PCR) with avian myeloblastosis virus reverse- transcriptase (AMV-RT). Incubation of the RT reaction at 46°C resulted in higher levels of amplified products as compared with the recommended 37°C. Preheating the reaction mixture at 55°C for 5 minutes further improved PCR yields. As an alternative to IC, PNRSV could be detected by RT-PCR directly in plant sap. Undiluted sap was negative in PCR, presumably due to the presence of PCR inhibitors. Dilution of sap (at least 1:50) resulted in a successful PCR amplification of the virus-specific DNA fragment. This direct procedure can be easily applied for large-scale testing, however, IC-PCR, which also avoids the inhibitors effect, proved to be more sensitive and suitable for low virus titers. | |
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