ISHS Acta Horticulturae 472: XVII International Symposium Virus and Virus-Like Diseases of Temperate Fruit Crops
FACTORS AFFECTING THE DETECTION OF APPLE STEM GROOVING VIRUS BY PCR ANALYSIS |
| Author: | D. James |
| Abstract:
Primers were identified which specifically amplify a 499 bp fragment in the coat protein coding region of apple stem grooving virus (ASGV) genome. These primers were used in polymerase chain reaction (PCR) analysis for the reliable detection of ASGV in Chenopodium quinoa, Nicotiana occidentalis, and in species of Malus and Pyrus. Isolates of ASGV in Malus and Pyrus from locations in Canada, China, Israel, Japan, Nepal, Pakistan, South Africa, and the U.S.A. were reliably detected in leaf and bark (budwood) tissue. Storage of the tissues at -80°C for more than 4 months did not affect the reliability of detection by PCR. A polyclonal antiserum was produced and ammonium sulphate purified IgG was used to develop an immunocapture RT-PCR. Triton-X was not necessary for the detection of ASGV, and it was also possible to combine the antibody incubation and virus sap incubation into a single step and still detect the virus. A procedure was developed where the virus was trapped without the use of any antibody (Tube Capture, or Tube Trapped Virus RT-PCR) facilitating the simultaneous detection of two different viruses. | |
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