2010年10月
Application of cDNA Macroarray for Simultaneous Detection of 12 Potato Viruses
PLANT DISEASE
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- ,
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- 巻
- 94
- 号
- 10
- 開始ページ
- 1248
- 終了ページ
- 1254
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1094/PDIS-12-09-0787
- 出版者・発行元
- AMER PHYTOPATHOLOGICAL SOC
A complementary DNA (cDNA) macroarray was developed for simultaneous detection of 12 different potato viruses. A suitable region in the viral genome for each was selected for Alfalfa mosaic virus, Cucumber mosaic virus, Potato aucuba mosaic virus, Potato leafroll virus, Potato mop-top virus, Potato virus A, Potato virus M, Potato virus S, Potato virus X, Potato virus Y, Tomato ringspot virus, and Tomato spotted wilt virus, and their respective cDNAs were cloned into plasmid vectors. Capture probes for each virus ranging from 290 to 577 bp were generated by polymerase chain reaction (PCR) and immobilized on a nylon membrane. Total RNAs were extracted from each of these virus infected-plants, and cDNAs were synthesized from the RNA extracts using a random 9-mer primer. Subsequently, PCR reactions were performed using one primer pair for each of the 12 viruses. During PCR, amplified cDNAs were labeled with biotin and used as a target for hybridization analyses on a macroarray membrane. Hybridization signals between capture probes for the 12 viruses and their respective target cDNAs were observed using chemiluminescent or colorimetric detection. In all viruses, hybridization signals with capture probes were detected only when homologous virus targets were examined, and no hybridization to healthy plant extract was observed, facilitating identification of each virus. The results by colorimetric detection agreed with those obtained using chemiluminescence. The macroarray method developed was 5 x 10(2) to 4 x 10(6) times more sensitive than enzyme-linked immunosorbent assay and 5 to 5 x 10(4) times more sensitive than reverse-transcription PCR, except for Alfalfa mosaic virus. Colorimetric detection and substantial reduction in cross-hybridization signals much improved the method compared with other array-based detection methods for practical use.
- リンク情報
- ID情報
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- DOI : 10.1094/PDIS-12-09-0787
- ISSN : 0191-2917
- CiNii Articles ID : 80021300611
- Web of Science ID : WOS:000282012900012