Development of Identification Chip for Simultaneous Detection of Multi-genera Plant Viruses and a Microsphere Suspension Array System for Rapid Detection of potyviruses

• Main Authors: Shu-Tsen Yeh, 葉書岑
• Other Authors: Ya-Chun Chang
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/34347238049418246214

碩士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 97 === Solanaceous plants contain a lot of important crops such as tobacco, tomato, potato, pepper and etc. In terms of the literatures, there are over 29 genera 95 species of plant viruses that can infect solanaceous crops in the world, and at least 13 virus species have been reported in Taiwan. The sensitive microarray techniques offer a great capability for parallel testing, and can be used to detect individual or multiple viruses in a single experiment. For this reason, we chose this method to detect and identify the important solanaceous plants-infecting RNA viruses. In Taiwan, RNA viruses frequently found in solanaceous crops include cucumoviruses, potyviruses and tobamoviruses. Therefore, we chose these three viral genera and the quarantined tombusviruses as the detection targets, and attempted to develop a multi-genera virus chip based on the plannar format. We separately designed degenerate primer pairs for each virus genus and the plant actin mRNA. Then we designed the oligonucleotide probes that allow identification of each virus to the species level from the region between the degenerate primer pairs. Finally, the probes were spotted onto a nylon membrane and immobilized by UV light. At present, one actin probe and 18 virus probes for four genera of plant viruses were designed. We used the equal molar mixture of the cDNA clones from four viral genera and the plant actin as the templates, and then optimized the concentration ratio of five degenerate primer pairs to overcome the difficulty of multiplex PCR. DIG-labeled targets were prepared from total RNA of the virus-infected plants by means of multiplex RT-PCR with five pairs of degenerate primers and dot-blot hybridization with the probes on the membrane. The detection sensitivities of multiplex RT-PCR and chip hybridization were 100 pg and 1 pg of RNA transcripts of Tomato bushy stunt virus, respectively. The results indicated that multi-genera virus chip could identify tombusvirus, cucumovirus, potyvirus and tobamovirus in single or mixed infection plant and seed samples correctly. In order to realize the most suitable storage condition for the chip, we tried to preserve the chip in different temperature condition, and took out the chip to hybridize with target once a month. The results showed that our chip could be kept for one year and still retained its detection ability, and its best storage condition was at -20oC. The non-plannar format of microsphere suspension array has been developed recently that might be high throughput and increased the speed of detection experiment. The target-specific primer extension (TSPE) of the system is suitable for microorganism detection. Therefore, we tried to develop a microsphere suspension array system that combines TSPE and microsphere hybridization for detection of solanaceous plant-infecting potyviruses. This TSPE of microsphere suspension array system could correctly identify five potyviruses in single or mixed infection samples.