ISHS Acta Horticulturae 472: XVII International Symposium Virus and Virus-Like Diseases of Temperate Fruit Crops
TWO RAPID EXTRACTION METHODS TO DETECT APPLE SCAR SKIN AND HOP STUNT VIROIDS BY RT-PCR |
| Authors: | H. Osaki, Y. Ohtsu, A. Kudo |
| Keywords: | apple scar skin viroid, hop stunt viroid, apple, Japanese pear, Japanese plum |
| Abstract:
Two simple protocols for plant tissue preparations suitable for the detection of apple scar skin viroid (ASSVd) and hop stunt viroid (HSVd) by the reverse transcription-polymerase chain reaction (RT-PCR) are described. The effect of the two protocols on the release of viroids from various tissues of apple, Japanese pear and Japanese plum, and the inhibition of RT-PCR in preparations by the two protocols were evaluated. In one protocol, a small amount of tissue was heated in 20 μl of distilled water for 10 min at 95°C and the supernatant was used for enzymatic amplification. In the other, a small amount of tissue was pressed by a vise with 20μl of distilled water and the squeeze was used for enzymatic amplification. Application of the two protocols to RT-PCR depended on the kinds of trees or tissues. In the two protocols, tissue homogenization, centrifugation, the use of organic solvents and ethanol precipitation are not required. These protocols can be used for the preparation of a large number of samples since they are simple and rapid. We were able to handle about 10 samples by heat or pressure treatment for RT-PCR in 30 minutes instead of 8 hours by previous methods. | |
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