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

茄科植物涵蓋多種重要經濟作物，據文獻記載全世界能感染茄科作物的病毒超過29屬95種，而台灣也將近有13種以上之病毒可感染茄科作物。由於田間的茄科作物常有複合感染的情形，而高靈敏度之生物晶片(microarray)具有強大的平行檢測(parallel testing)能力，可在單一實驗中同時檢測多種目標病毒，因此被我們選擇作為診斷與鑑定茄科重要病毒的平台。在台灣危害茄科作物的RNA病毒以Tobamovirus、Potyvirus和Cucumovirus三屬為主，因此我們以這三屬病毒為檢測對象，同時將台灣尚未有報告之檢疫病毒Tombusvirus屬病毒納入晶片中，設計出以平面系統為基礎的多屬植物病毒鑑定晶片。針對這四屬病毒與植物actin mRNA，先分別設計各自的廣效性引子對，並在廣效性引子對之間選取序列差異性最高之區域，設計各種病毒的專一性寡核苷酸探針。而後將廠商合成的探針點漬於尼龍膜上，以UV光照射固定後備用。目前晶片所涵蓋的病毒探針種類已達四屬十八種。接著利用四屬病毒以及植物actin之cDNA株，依等莫耳數混合後作為模板，藉由multiplex PCR反應測試五組廣效性引子對以不同比例混合時之擴增效果，藉此調整各廣效性引子對之比例，以建立multiplex PCR最佳的擴增條件。標的物之製備方式為以植物全RNA為材料，利用五組廣效性引子對進行multiplex RT-PCR，並在擴增過程中以DIG標定，得到DIG標定之產物作為標的物，再與晶片進行逆墨點雜合反應，即可由雜合訊號判讀病毒檢測結果。經由我們調整後的實驗條件進行反應，multiplex RT-PCR與多屬植物病毒鑑定晶片之檢測靈敏度分別為100 pg與1 pg之Tomato bushy stunt virus RNA轉錄體。實驗結果顯示多屬植物病毒鑑定晶片可正確地檢測出此四屬病毒單獨或複合感染的茄科植物與種子樣品。為了解多屬植物病毒鑑定晶片之最適保存條件，將所製備的晶片分別乾燥保存於不同溫度條件下，每個月定時取出進行雜合反應測試，結果顯示此晶片可保存一年仍具有檢測功能，並且其最適存放環境為-20oC冰櫃。生物晶片依其基質的變化可分為平面系統(planar array)以及非平面系統(nonplanar array)兩種形式，而非平面系統之流式微珠陣列相較於傳統平面系統具有更便利快速之優勢，而流式微珠陣列系統中以target-specific primer extension (TSPE)方法較為彈性而簡單。因此我們以流式微珠陣列系統之TSPE方法為基礎，研發可同時快速檢測感染重要茄科作物的六種potyviruses之檢測平台，並以單獨或複合感染之potyvirus植物樣品進行測試，結果顯示出流式微珠陣列之TSPE系統可正確地檢測出健康植物以及五種potyvirus單獨感染之植物樣品，亦可準確地檢測出potyvirus複合感染之樣品。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.口試委員會審定書..............................................................................Ⅰ謝.........................................................................................Ⅱ要 ....................................................................................... IIIbstract.................................................................................... V、前人研究 ................................................................................ 1.1 植物病毒危害茄科作物之情形 .............................................................. 1.2 生物晶片................................................................................ 3.3 Tombusvirus屬病毒之介紹 ................................................................ 7.4 Cucumovirus屬病毒之介紹 ................................................................ 9.5 Potyvirus屬病毒之介紹 .................................................................. 9.6 Tobamovirus屬病毒之介紹 ................................................................ 10、材料與方法............................................................................... 12.1 材料 .................................................................................. 12.1.1實驗植物與栽種方式 ..................................................................... 12.1.2實驗病毒 .............................................................................. 12.2 方法................................................................................... 13.2.1 多屬植物病毒鑑定晶片之實驗方法 ......................................................... 131) 病毒屬專一之廣效性引子對與植物actin mRNA引子對之設計 ...................................... 132) 種專一之核酸探針設計與製備 ............................................................... 143) 植物全RNA萃取 (Plant Total RNA Extraction)............................................... 144) 反轉錄反應 (Reverse Transcription, RT) .................................................. 155) 聚合酶連鎖反應 (Polymerase Chain Reation, PCR) .......................................... 16.2.2 多對廣效性引子對聚合酶連鎖反應(multiplex PCR)最佳條件測試) .............................. 161) cDNA株製備 (cDNA Clone Preparation) ................................................... 162) 轉形試驗 (Transformation) .............................................................. 173) 轉形株之篩選 ............................................................... ............174) 質體之小量製備 (Minipreparation)......................................................... 175)多對廣效性引子對聚合酶連鎖反應之最佳引子對比例之測試 (Multiplex PCR Primer Ratio Test) ...... 186) 多對引子反轉錄聚合酶連鎖反應 (Multiplex RT-PCR)........................................... 19.2.3 Multiplex RT-PCR與多屬植物病毒鑑定晶片之靈敏度分析 ..................................... 191) 限制酶直線化質體 (Plasmid Linearization) ................................................ 192) 胞外轉錄反應 (In Vitro Transcription) ................................................... 203) 標的物之製備 (Target Preparation) ....................................................... 204) 雜合反應 (Hybridization) ................................................................ 215) 靈敏度測試 (Sensitivity Test) ........................................................... 22.2.4 多屬植物病毒鑑定晶片之最佳保存條件測試 .................................................. 22.2.5 流式微珠陣列系統 (Microsphere Suspension Array System) ................................ 221) 標的物專一性引子之設計 ................................................................... 222) Anti-tag序列之挑選 ..................................................................... 233) 於微珠上鍵結anti-tag序列 (Coupling Anti-tag Oligonucleotides to Microspheres) ............ 234) Potyvirus之多對引子反轉錄聚合酶連鎖反應 (Multiplex RT-PCR) ............................... 245) 標的物專一性引子延伸法 (Target-Specific Primer Extension, TSPE)............................ 256) 雜合反應與檢測 (Hybridization and Detection) ............................................. 25、結果 .................................................................................... 27.1 Tobamovirus、potyvirus、cucumovirus與tombusvirus於茄科植物上引起之病徵 ................... 27.2 以RT-PCR反應測試五組廣效性引子對之專一性 ................................................. 27.3 Multiplex PCR之最佳引子對比例之測試 ..................................................... 28.4 Multiplex RT-PCR可正確擴增出植物樣品所含之目標病毒 ....................................... 29.5 Multiplex RT-PCR之靈敏度測試 ........................................................... 29.6 多屬植物病毒鑑定晶片之靈敏度測試 ........................................................ 30.7 多屬植物病毒鑑定晶片可準確鑑定出tombusvirus感染之植物樣品 ................................ 30.8 多屬植物病毒鑑定晶片可準確鑑定出不同屬病毒複合感染之植物樣品................................ 31.9 多屬植物病毒鑑定晶片可正確檢測出單獨或複合感染之茄科種子樣品............................... 31.10 多屬植物病毒鑑定晶片之最適儲存條件之測試 ............................................. 32.11 流式微珠陣列系統可專一檢測5種potyvirus............................................... 33.12 流式微珠陣列系統可正確鑑定出potyvirus複合感染樣品..................................... 33、討論.................................................................................... 34、參考文獻................................................................................ 39、表 .................................................................................... 46、圖 .................................................................................... 5