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  • 學位論文

可同時檢測三種病毒之香蕉生物晶片系統之開發

Development of a biochip system for simultaneous detection of three different banana viruses

指導教授 : 張清安
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摘要


香蕉為台灣最具經濟重要性之果樹,自五零年代起每年均穩定外銷創造可觀之產值。香蕉之栽培容易受到各種病害的危害,導致產量與品質的損失。特別是幾種能夠造成系統性感染之病害,包括鐮刀菌引起之黃葉病及幾種病毒病害,會藉由帶病親本垂直感染,防治難度高,對香蕉之栽培威脅最大。已知的病毒病害中香蕉萎縮病毒(Banana bunchy top virus, BBTV)、胡瓜嵌紋病毒(Cucumber mosaic virus, CMV)及香蕉苞葉嵌紋病毒(Banana bract mosaic virus, BBrMV)都可以藉由組織培養過程而全面垂直傳染至後代種苗,因此此等病害之防除必須在組培繁殖前檢定其種原,確認無病毒後方能進行複製繁殖。過去產業界通常利用傳統之 ELISA 或 PCR 技術執行香蕉病毒檢測,但因為有多重病毒感染之疑慮,必須分別進行多次單一病毒之檢測,時間與成本並不經濟。故本研究嘗試研發生物晶片檢測系統,期於單次檢測流程中即可完成多種病毒的檢測,以節省成本提升檢測效率。本研究室已完成 CMV 與 potyvirus 之生物晶片檢測系統,本研究將針對 BBTV 研發其生物晶片,並整合構築能夠同時檢測上述三種病毒之生物晶片系統。由於台灣尚未發生 BBrMV,故本研究目前所為之檢測試驗均以與 BBrMV 同屬 potyvirus 屬之蕪菁嵌紋病毒 (Turnip mosaic virus, TuMV)作為測試標的進行模擬,並且在設計 PCR 引子時乃以可廣效性增幅所有 potyviruses 之策略進行。目前測試結果已確認引子對 C511 可穩定檢測各種不同胡瓜嵌紋病毒分離株,獲得 511 bp 大小之 DNA 產物。而 P374 引子對則可穩定增幅至少 20 種不同的 potyviruses,獲得一個 374 bp 之增幅產物。針對 BBTV 則分別就其 DNA 1 與 DNA 3 基因區域分別設計 B313 及 B251 二組引子對,結果均證實分別可以獲得 313 bp 與 251 bp 之增幅產物。但後續針對上述 PCR 產物之序列,設計專一性探針並進行晶片雜合反應測試時,發現針對 B251 產物序列所設計的超過十組以上探針均因會產生非特異性雜合反應而不適合後續晶片系統之應用。但針對 313 bp 產物序列所設計之二組探針(B-pb-2 及 B-pb-3)則可產生理想之雜合反應。另外針對 CMV 與 BBrMV 之增幅產物則分別選出兩個專一性探針(Cpb-2 與 Ppb-ci2)可獲得理想的雜合反應。測試所選出的三組引子對證實可以組合成為多目標型(multiplex RT-PCR)增幅系統,且增幅之產物可以與所選出的各組探針產生專一性雜合反應,但不與非對應之探針發生非專一性雜合。且針對上述三種病毒單獨或複合感染之情況下,此一 multiplex 系統均能有效將單獨或複合感染病毒檢測出來。對於無病毒感染之健康香蕉或組培苗樣品均不產生非專一性增幅產物,或於晶片雜合反應中出現非專一性訊號。此一檢測系統除可應用於田間香蕉植株樣品之檢測外,也適用於組織培養苗之病毒檢測。面對台灣各地所採集之20株以上之不同 BBTV 與 CMV 病毒分離株,此系統亦均能獲得穩定無差異之檢測結果。由於 BBTV 屬於 DNA 病毒與 CMV 及 potyviruses 之 RNA 病毒不同。傳統上進行 此二病毒之多目標增幅時香蕉組織樣品必須分別以DNA 與 RNA 套組純化總量核酸,流程較為繁複且成本較高。本研究已證實應用過去文獻已報導之緩衝溶液與快速組織萃取流程,可適用於本檢測系統同時穩定檢測出 BBTV 及 CMV 兩種不同基因體病毒。此一簡速流程與傳統分別應用DNA 與 RNA 核酸純化套組以萃取全量核酸之病毒檢測結果並無差異。另外我們也發現利用一種商用磁珠套組亦可同時純化香蕉組織之全量 DNA 與RNA 樣品並且獲得無差異之多目標型病毒增幅與晶片探針雜合結果。經由此等全量核酸萃取流程,確實可在不影響最終晶片檢測的結果下,提升檢測效率降低成本。

關鍵字

香蕉 植物病毒 生物晶片

並列摘要


Banana is ranked the most economically important fruit crops in Taiwan. It is exported consistently to foreign countries and evident output value has been created annually since 1950s. During cultivation, banana was always suffered to various diseases and resulted significant loss on yield and quality. Among the diseases that threaten banana’s cultivation, those causing systemic infection such as Panama and virus diseases are the most devastating ones due to the difficulty of control and easiness to transmit from infected mother plants to young progenies. Banana viruses include Cucumber mosaic virus (CMV), Banana bunchy top virus (BBTV) and Banana bract mosaic virus (BBrMV) can vertically infect all tissue culture propagated growing materials, therefore banana mother plants need to be indexed for virus infection before tissue culture cloning process. Traditionally banana industry applied ELISA or PCR-based techniques to detect banana viruses. Due to multiple virus infection possibility, banana mother stocks should separately indexed by different virus probes or techniques. This process is vital but time and labor consuming and also cost ineffective. The purpose of this research is to develop a biochip DNA microarray system that can detect all three banana viruses simultaneously so that the efficiency of virus indexing will be increased and therefore the cost can be reduced. Currently we have successfully designed and selected three pairs of PCR primers respectively specific to CMV, BBTV and BBrMV. Due to BBrMV is now considered as quarantine pest in Taiwan, its live cultures are not available for testing. Therefore, we took an approach by designing a Potyvirus genera specific PCR primer pair that could amplify at least more than 20 different potyvirus species, so that the amplification spectrum could theoretically cover BBrMV. During testing period, we consistently used another potyvirus, Turnip mosaic virus (TuMV), as model to test the feasibility of the detection system. Our result showed the primer pair, namely C511, could specifically amplify more than 10 CMV isolates collecting from different crops including banana and resulted a 511 bp PCR product. The primer pair, P374, could successfully amplify at least 20 different potyviruses and all producing a 374 bp amplicon. As for BBTV, we designed and selected two primer pair, i.e. B313 and B251, specific respectively to sequences in DNA 1 and 3 of the virus that could obtain respectively 313 bp and 251 bp PCR products. However in the next stage when oligonucleotide probes were designed to hybridize with their corresponding amplicon, more than 10 probes designed from the 251 bp amplicon sequences were unsuccessful for hybridization. Therefore, the further testing of BBTV detection by primer pair B251 was terminated. Fortunately, two probes, Bpb-2 and Bpb-3, designed for capturing the 313 bp amplicon were found highly dependable, therefore they were chosen to combine with another two oligonucleotide probes (Cpb-2 and Ppb-ci2) specific respectively to CMV and BBrMV into an integrated DNA microarray system to hybridize with PCR products amplified by multiplex primer pairs of C511, P374 and B313 mixture. Our result showed that these four selected oligo-probes when immobilized on biochip could consistently react with its corresponding amplicon specifically and gave no non-specific signals against each other. Using this multiplex detection microarray system, we were able to demonstrate either single or mixed infection of these three banana viruses could consistently be detected and differentiated. Not only field collected banana plants but also small plantlets during tissue culture stage could be used as targets in this detection system. Moreover, the system is highly specific that no signal was resulted in reaction to healthy uninfected banana plant samples and tissue cultured plantlets. In this study we have tested more than twenty isolates each of BBTV and CMV and found all isolates were uniformly detected by the system. Furthermore, due to BBTV is a DNA virus different from the RNA genome of CMV and BBrMV, the preparation of total nucleic acids from sample tissue in the first step of PCR reaction is traditionally different. In the beginning of our experiment, we always applied separate commercial DNA or RNA nucleic acid purification kits to extract DNA from BBTV and RNAs from CMV or BBrMV infected tissues. These separate procedures were apparently costly and labor intensive. In order to reduce the cost and increase efficiency, we tested the possibility of using a literature cited buffer and extraction protocol for quick extraction of both RNA and DNA from plant tissue before PCR amplification. The result indicated that the same buffer and extraction procedure was feasible in our multiplex banana virus detection system as a one-step total nucleic acid extraction before PCR. We also found that a commercial total nucleic acid (DNA plus RNA) purification system using magnetic beads could also be applied in our detection system for one-step nucleic acid extraction. There was no difference found in the final biochip result between either the two quick extraction systems or those traditional extraction protocols.

並列關鍵字

Biochip Plant virus Banana

參考文獻


楊淑惠,「由薄葉牛皮消(Cynanchum taiwanianum Yamazaki)所分離之胡瓜嵌紋病毒特性研究」,碩士論文,國立屏東科技大學植物保護研究所,屏東(2008)。
何宇倫,「茄科重要病毒鑑定晶片之研發與應用」,碩士論文,國立台灣大學植物病理與微生物學研究所,台北(2007)。
張馨元,「香蕉萎縮病毒及香蕉條紋病毒甲基化之分析與比較」,碩士論文,臺灣大學植物病理與微生物學研究所,台北(2011)。
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被引用紀錄


黃莉真(2014)。可同時檢測多種百香果病毒之多目標型增幅及晶片檢測系統〔碩士論文,朝陽科技大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0078-2611201410181994
張至超(2017)。馬鈴薯病毒多目標型檢測系統之改良及一種新紀錄之扁蒲種傳病毒之分子特性分析〔碩士論文,朝陽科技大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0078-2712201714433463
賴品娟(2017)。台灣常見消費肉品之晶片快速檢鑑技術之開發〔碩士論文,朝陽科技大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0078-2712201714433362

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