1992 Fiscal Year Final Research Report Summary
Application of polymerase chain reaction in highly sensitive gene diagnosis of plant viruses and viroids.
| Project/Area Number |
02556007
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
植物保護
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
SHIKATA Eishiro Hokkaido Univ., Fac.Agr., Prof.Emeritus, 名誉教授 (00001389)
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| Co-Investigator(Kenkyū-buntansha) |
SUGIMOTO Noritaka Yuki Gosei Kogyo Co., LTD, Lab.Chief, 生化学研究室, 室長
OHSHIMA Kzusato Saga Univ., Fac.Agr., Assistant, 農学部, 助手 (00176869)
UYEDA Ichiro Hokkaido Univ., Fac.Agr., Assoc. Prof., 農学部, 助教授 (10113523)
KOJIMA Makoto Niigata Univ., Fac.Agr., Prof., 農学部, 教授 (00001454)
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| Project Period (FY) |
1990 – 1992
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| Keywords | Polymerase chain reaction / Gene diagnosis / Plant viruses / Nucleotide sequence / Microplate Hybridization |
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| Research Abstract |
1) Nucleotide sequences of plant viruses and viroids were analyzed and primers for PCR were designed. Analyzed plant viruses and viroids were rice dwarf virus, rice black-streaked virus, rice ragged stunt virus, potato virus Y, turnip mosaic virus, potato leafroll virus, soybean mosaic virus, bean yellow mosaic virus, chrisanthemum stunt viroid, hop latent viroid, several isolates of hop stunt viroid.
2) Extraction methods of viral RNAs and viroids were developed and these RNAs were successfully used for cDNA synthesis for PCR template.
3) Detection of viroids by PCR was found to be more sensitive than by conventional electrophoresis and hybridization methods.
4) Detection of viruses by PCR was found to be more sensitive than by conventional ELISA and hybridization methods.
5) Microplate hybridization method, where PCR-amplified DNAs were hybridized with a non-radioactive probe in solution, was developed. The method was 10 times higher in detection sensitivity than PCR alone. And the fact that it confirms the sequence identity by hybridization simultaneously makes the method superior to PCR alone.
6) Contamination with viroid RNAs was encountered during PCR detection. It was found that the first extraction step was the most critical preventing the contamination.
7) The PCR method successfully detected introduced genes in a transgenic plants. The method was rapid and simple and thus superior to a conventional southern blot hybridization.
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