Development of Identification Chip for Important Solanaceous Plant Viruses

• Main Authors: Yu-Lun Ho, 何宇倫
• Other Authors: 張雅君
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/06025076191105758234

碩士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 95 === Solanaceous plants include many important vegetables and fruits such as tomato, pepper, potato, etc. There are many kinds of plant viruses which can infect solanaceous plants and mixed infection happens frequently in the field. According to the literatures, more than 20 genera 80 species viruses infect solanaceous plant. In Taiwan, there are over 40 viruses including tobamoviruses, potyviruses and cucumoviruses reported to infect solanaceous plants and cause heavy losses. ELISA and RT-PCR are commonly used methods for plant virus detection. Nevertheless, it will be preferable to have a specific and sensitive method which can detect many viruses simultaneously. Therefore, a solanaceous virus chip based on the principle of DNA microarray was attempted to develop. At first we download viral amino acid sequences from GenBank. After using ClustalX software to align amino acid sequences, we used CODEHOP software to separately design degenerate primers of potyviruses, tobamoviruses and cucumoviruses from aligned amino acid sequences. After that, species-specific oligonucleotide probes were designed from the regions between each degenerate primer pair. DIG-labeled or Cy3-labeled targets were prepared from total RNA of infected tissues by means of multiplex RT-PCR with all sets of degenerate primers. Oligonucleotide probes were immobilized onto nylon membrane or slide chip, hybridized with the targets and then the hybridization signals were detected. The results indicated that solanaceous virus chip could correctly identify potyviruses, tobamoviruses and cucumoviruses in single and mixed infection plants. Up to the present, we have designed 14 virus probes for three genera of plant viruses. This virus chip is expandable to include new probes which are required for particular purpose.