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Acta Horticulturae


Krisbai, L., Plant Health and Soil Conservation Station, Budapest, Hungary
Kölber, M., Plant Health and Soil Conservation Station, Budapest, Hungary

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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Evaluating the use of immunocapture and sap-dilution PCR for the detection of prunus necrotic ringspot virus
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Krisbai, L., Plant Health and Soil Conservation Station, Budapest, Hungary
Kölber, M., Plant Health and Soil Conservation Station, Budapest, Hungary

Evaluating the use of immunocapture and sap-dilution PCR for the detection of prunus necrotic ringspot virus
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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