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  • 學位論文

蝴蝶蘭病毒檢測用之核酸快速簡易萃取及黃葉病菌分子鑑定技術之開發

Development of techniques for quick and easy RNA extraction for virus detection and molecular identification of yellow leaf pathogens of phalaenopsis orchids

指導教授 : 陳金枝 張清安
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摘要


蝴蝶蘭是我國重要的外銷花卉產業之ㄧ,近年來荷蘭進入國際市場角逐,並以推廣無病毒健康苗為賣點強力促銷,對我國產品構成威脅。因此加強生產流程中之病毒病管控,強化種苗之品質,乃維護我國蘭花產業競爭力之重要環節,而病毒檢測更是管控蘭花病毒病之關鍵技術。目前已知可以感染蘭花的病毒種類有59種,其中又以蕙蘭嵌紋病毒Cymbidium mosaic virus (簡稱CymMV)、齒舌蘭輪斑病毒Odontoglossum ringspot virus(簡稱ORSV)影響最為嚴重。進行病毒核酸檢測時,第一道程序必須由植物組織進行全量核酸純化,傳統上慣常使用核酸萃取純化試劑組,但當面對大量的樣品檢測時,應用試劑套組所耗費的時間及成本均高。本研究希望能開發核酸快速萃取法,簡化處理流程,在不影響檢測準確度前提下,達到降低成本及增加檢測效率的目的。研究初期應用國外已報告的簡易核酸萃取方法(Thomonas et al., 1995),利用其 TPS緩衝液配方,針對感染蝴蝶蘭之 ORSV 及 CymMV 進行核酸快速萃取模式之建立,將植株葉片罹病組織與 TPS 緩衝液以 1:4 (g/ml) 比例研磨均勻後,經 80℃處理 10 分鐘,萃出液再經無菌水稀釋 10 倍後進行單步驟RT-PCR反應可成功檢出ORSV及CymMV。應用相同模式於感染蝴蝶蘭之Tospoviruses 屬病毒 CaCV (Capsicum chlorosis virus )之檢測,於 60℃,熱處理10分鐘後即可順利取得病毒核酸用於RT-PCR的病毒檢測。本研究結果顯示核酸快速萃取法應用於植物病毒核酸的萃取,進而作為植物病毒之核酸檢測,具有相當大的潛力。另外我們也比較了TPS及其他六種不同的緩衝液之萃取與檢測效果,結果發現本研究所配製的CarTPS 緩衝液檢測結果優於文獻上的TPS。不過這二種快速萃取的檢測結果與運用試劑套組萃取法相較仍較不穩定,在部分病毒濃度較低的樣品中快速萃取法則無法獲得理想的PCR增幅。反之,以試劑套組萃取之核酸則可獲得穩定之增幅。評估認為此一快速萃取技術仍可做為大量蘭花樣品之初級篩選,也適用於病毒種類之快速鑑定。 本研究另一個目標為發展可鑑別蝴蝶蘭黃葉病病原之分子檢測技術。目前文獻記載有Fusarium solani (Fs) 與F. oxysporum (Fo)兩種鐮胞菌可能是引起蝴蝶蘭黃葉病的病原,由於此兩種真菌之發病生態迥異,若未能精確加以鑑定將無法採取正確之防治措施以為因應。因此本研究希望能開發出可以快速鑑別此二病原菌之方法。研究中針對不同來源之蘭花黃葉病菌,利用逢機核酸多型性分析(RAPD)之策略,篩選出編號OPAT-A02 之引子所增幅出的幾個核酸多型性片段具有區別Fs 與Fo的效果。針對此鑑別性核酸片段進行選殖定序,再就其序列設計出可專一性增幅Fs菌株的引子對FS239及增幅Fo菌株的引子對FO520,二者可分別增幅出239 bp及520 bp之核酸產物。將此二鑑別性引子對於分離自蝴蝶蘭及不同作物之27種Fs與分離自國蘭及不同作物之16 種Fo菌株進行PCR反應測試,結果證實二者均具有專一性的增幅與鑑別效果。進一步測試此二引子對與蘭花上常見的真菌病原如疫病菌 ( Phytophthora parasitica Dastur )、炭疽病菌( Colletotrichum gloeosporioides Penzig )、灰黴病菌( Botrytis cinerea )及白絹病菌( Sclerotium rolfsii Sacc )之反應,結果皆無任何增幅訊號產生。不過此二引子對目前仍無法鑑別Fusarium 之不同生理小種以及區分病原與非病原性菌株。實驗結果發現利用FS239引子對進行Fs菌株基因體核酸之偵測時,其靈敏度可達1 ng/μl;而FO520引子對之靈敏度達10 pg/μl。應用此二鑑別性引子對於呈現黃葉病徵之蝴蝶蘭樣品直接進行PCR檢測時,發現FS239引子對可以由表面消毒的病株根部、莖基部及葉基部組織上順利增幅出Fs的專一性239 bp片段,其檢出率與傳統組織分離法檢出Fs之比率相符甚至更高。我們更發現若將蝴蝶蘭黃葉病患部組織經表面消毒後,置於水瓊脂培養皿內培養三天,待組織塊上長出菌絲後再取出萃取全量核酸以FS239引子對進行PCR檢測,可以獲得與需時七天以上的傳統組織分離法相符或更高的檢出比率。此等結果證實FS239引子對確實可以應用於輔助傳統組織分離法之病害診斷工作,具有提升效率與檢出率之效果。

並列摘要


Phalaenopsis is currently the most important exporting item for the ornamental industry in Taiwan. In recent years, companies from the Netherlands have been taking advantage of their virus-free planting materials propagation system to promote their phalaenopsis; that apparently has threaten the competitiveness of Taiwan’s orchid industry in the international market. Therefore, improving the quality and virus control status of our phalaenopsis products has become the key issue for maintaining the competitiveness of our orchid industry, and for this reason, a decent virus detection technology is inevitable to accomplish this goal. Fifty-nine viruses have been known to infected orchids. The Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are recognized as the most widespread and economically important viruses for orchid industry worldwide. For plant RNA viruses, development of a RT-PCR assay would provide a significant improvement over current detection technology. Nucleotide sequences for the design of PCR primer are readily available for many viruses but nucleic acid extraction from plant tissues is a laborious and time-consuming step in RT-PCR procedures. The TPS buffer have been reported to apply on quickly release virus nucleic acid from plant tissue for detection (Thomson et al. 1995). In this study, we demonstrated an applicable quick and easy protocol for the extraction of total RNA from phalaenopsis orchid sample before amplification of ORSV and CymMV in RT-PCR. In this protocol, virus-infected leaves were groud 1:4 (g/ml) in TPS buffer, the resulting crude saps were incubated at 80℃ for 10 min, diluted 10 fold with sterilized H2O and subsequently used as RNA template in RT-PCR. Similar procedure with a lower (60℃) temperature treatement was found to be feasible for the detection of Capsicum chlorosis virus, (CaCV). We also tested the feasibility of some potential buffers and found that a CarTPS buffer is comparable to TPS buffer for the extraction of plant total RNA with decent quality for RT-PCR. However, after careful and repetitive comparison tests it was realized that our developed quick and easy RNA extraction protocol could not detect virus infection on certain orchid samples especially when they accumulated minor virus concentration. These samples could only be detected by purifying the total RNA for RT-PCR by the use of commercialized plant total RNA extraction column. Nevertheless, our protocol was confirmed in this study to be applicable for the identification of viruses in samples with normal accumulation of viruses. Another goal in our study is to develop the molecular method for the differential identification of Fusarium fungus infecting phalaenopsis orchids. Fusarium solani and F. oxysporium have been reported to infect orchidaceous plant causing yellow leaf and wilting diseases. Traditionally, Fusarium spp are difficult to be identified because of their diversified morphological characteristics and virulence. For this reason, in this studies are attempted to test the feasibility of using molecular techniques to conduct a fast and precise differentiation and identification of F. solani and F. oxysporium by the use of random amplification of polymorphic DNA (RAPD), which is one of the powerful molecular tools for fungal pathogen identification. Upon testing varius RAPD primers, the OPA02 was found to amplify differentially F. solani and F. oxysporium producing specific DNA fragments the the genomic DNA of F. solani and F. oxysporium isolated from orchid. The differentially amplified specific DNA fragments was then recovered, cloned and sequenced. Furthermore the specific primer set FS239 and FO520 were designed to the known nucleotide sequents of the special DNA fragment of F. solani and F. oxysporium, respectively. The predicted 239 bp and 520 bp DNA fragment were amplified from the primer set of FS239 and FO520, respectively. Therefore, The two specific DNA fragment were not amplified in the genomic DNA of the Phytophthora parasitica Dastur,Colletotrichum gloeosporioides Penzig, Botrytis cinerea, Sclerotium rolfsii Sacc. but were amplification some different formas speciales isolates of F. solani and F. oxysporium. The results showed that the two specific primer set developed can successfully detect F. solani spp and F. oxysporium spp genomic DNA for PCR. And used FS239 primer that the minimal DNA concentration could be detected is about 10 ng/ml; used FO520 primer could be detected is about 10 pg/ml. FS239 and FO520, was used to test naturally infected orchid tissue collected from the field. It appeared that the FS239 and FO520 DNA fragment could easily be amplified in gDNA of symptomatic orchid pseudostems, root and leaf. The primer set FS239 and FO520 can be efficiency in auxiliary identifying F. solani and F. oxysporium and accurately and rapidly detecting diseases caused in the field.

參考文獻


張清安,蘭花種苗病毒檢測與相關產業發展趨勢,農業生技產業季刊
張清安,我國蘭花病毒病害防治之策略、歷程與未來展
黃偉洲,海芋嵌紋病毒對彩色海芋生長之影響及其分子檢測系統之建
張清安,蘭花病毒病,植物保護技術專刊系列 1-行政院農業委員會
檢疫病害診斷鑑定技術研習會專刊-行政院農業委員會動植物防

被引用紀錄


黃尚仁(2012)。可同時檢測三種病毒之香蕉生物晶片系統之開發〔碩士論文,朝陽科技大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0078-1511201214173561

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