臺灣博碩士論文加值系統

水稻紋枯病菌 (Rhizoctonia solani) 會造成水稻紋枯病 (sheath blight of rice)。此病原菌為土傳性病原菌，能以菌核及菌絲殘存於土壤中，可自莖基部侵入水稻植株並於葉鞘部大量繁殖，進而造成水稻發生枯萎的虎斑狀病徵，為全球水稻產業上一大重要限制因子。為了預防此病害發生造成水稻產業上的經濟損失，發展一個具備專一、快速、且能夠現地進行檢測之診斷工具用以檢測田間之R. solani，就此病害管理而言是重要策略之一。本研究擬開發一個水稻紋枯病菌之現地檢測技術，用以檢測R. solani。根據試驗結果，本研究已開發出快速萃取試劑，可用於田間檢體之核酸萃取，並完成專一性引子對SMS RS1-F/ SMS RS1-R及GMRS-3/ITS1靈敏度及專一性測試。本研究以四種不同萃取系統進行比較，結果顯示自動萃取系統搭配專一性引子對SMS RS1-F/ SMS RS1-R之檢出率為100%，為四種萃取系統中最穩定；而使用快速萃取試劑次之，其檢出率為96%，此結果證實本研究所開發的快速萃取試劑確實能有效地用於檢測紋枯病。透過同日間分析 (intraday assay) 及異日間分析 (interday assay) 測試結果，證實本研究所開發的不同萃取系統皆能夠穩定地進行水稻檢體的核酸萃取。以不同病程之水稻檢體進行萃取之in-planta試驗結果顯示，以自動萃取系統進行檢體核酸萃取可獲得較穩定的檢出率，平均檢出率高於80%。未來擬進一步針對田間帶菌土壤進行萃取測試，使此檢測系統更能落實於水稻田間紋枯病菌之現地檢測工作中。

Sheath blight of rice (ShB) caused by Rhizoctonia solani is a major limiting factor to rice production. This soil-borne fungal pathogen can survive in the soil by sclerotia and mycelia. They are the primary inoculum in rice fields. The primary inoculum has ability to infect the rice sheath from the stem base and multiply in the tissues causing lesion symptoms on sheath. In order to prevent the outbreak of rice sheath blight and to reduce the economic loss of rice yield, it’s critically important to develop a rapid detection method with specific, fast, and on-site advantages for disease management. In this study, the rapid extraction reagent which can be used for on-site detection was developed. The specificity and sensitivity of the novel primers set SMS RS1-F/SMS RS1-R and the reference-primer set GMRS-3/ITS1 were tested. In addition, this study developed the four different extraction protocols for rice sheath infected by R. solani. Moreover, the intraday- and interday-assays were performed for the reproducibility evaluation of the detection methods developed in this study. The results of reproducibility evaluation indicated that all developed detection protocols were suitable to be used for diseased rice sheath. In the future, an on-site will be developed for field soil to make the detection system to be more completely.

目錄
中文摘要.................................................I
Abstract..............................................III
謝誌....................................................V
目錄...................................................VI
圖表目錄................................................IX
壹、前言.................................................1
貳、文獻回顧.............................................3
一、水稻紋枯病菌之簡介....................................3
(一) 水稻疑似紋枯病 (Brown sheath blight) 介紹............4
二、水稻紋枯病菌之鑑定與檢測方法及技術開發之現況............5
(一) 傳統植物病理研究法..................................5
(二) 分子生物檢測技術....................................5
三、即時定量聚合酶連鎖反應................................6
四、DNA萃取的方法與試劑開發現況...........................7
五、研究目的.............................................8
六、論文架構.............................................9
參、材料與方法..........................................10
一、 Rhizoctonia solani之專一性引子對與探針設計..........10
二、 田間不同罹病程度水稻樣本之分子檢測...................10
三、供試菌株分離培養與鑑定...............................10
(一) 供試菌株之採集與分離培養............................10
(二) 供試菌株之鑑定.....................................11
1. 核酸萃取.............................................11
2. 核酸擴增與電泳分析...................................12
3. 定序分析.............................................13
三、 即時定量聚合酶連鎖反應分析...........................13
(一) SYBR green-based real-time PCR....................13
(二) TaqMan probe-based real-time PCR..................14
四、 專一性引子靈敏度分析................................14
(一) 一般PCR靈敏度測試..................................14
(二) SYBR green-based real-time PCR靈敏度測試...........15
(三) TaqMan probe-based real-time PCR系統靈敏度測試.....15
五、 水稻紋枯病帶菌檢體之核酸萃取.........................15
(一) 單一試劑萃取系統 (one buffer extraction)...........15
(二) 自動萃取系統 (taco™ mini automatic nucleic acid extraction system, Genereach, Taichung, Taiwan)........16
(三) 管柱萃取純化法 (column purification)...............16
(四) 有機溶劑萃取純化法 (organic sorvant reagents purification)..........................................16
六、 核酸萃取再現性測試..................................17
肆、研究結果............................................18
一、水稻紋枯病分子檢測系統之建立..........................18
(一) 水稻紋枯病菌引子專一性測試..........................18
(二) 水稻紋枯病菌引子對之靈敏度測試.......................18
二、水稻快速萃取試劑之開發與測試..........................20
(一) 水稻快速萃取試劑之開發..............................20
(二) 水稻快速萃取試劑之測試..............................20
三、不同萃取系統萃取效果之評估............................21
(一) 單一試劑萃取系統 (one buffer extraction)...........21
(二) 自動萃取系統 (taco™ mini automatic nucleic acid extraction system, Genereach, Taichung, Taiwan)........22
(三) 管柱萃取純化法 (column purification)...............22
(四) 有機溶劑萃取純化法 (organic sorvant reagents purification)..........................................23
四、不同萃取系統之檢測再現性評估..........................23
(一) 單一試劑萃取系統 (one buffer extraction)...........23
(二) 自動萃取系統 (taco™ mini automatic nucleic acid extraction system, Genereach, Taichung, Taiwan)........24
(三) 管柱萃取純化法 (column purification)...............24
五、不同帶病程度之水稻萃取測試............................25
(一) 單一試劑萃取系統 (one buffer extraction)...........25
(二) 核酸自動萃取系統 (taco™ mini automatic nucleic acid extraction system).....................................26
伍、討論................................................27
一、專一性引子設計......................................27
二、快速萃取試劑之開發及測試.............................27
三、不同萃取系統萃取效果及成本分析評估之結果...............28
四、不同萃取系統之萃取再現性評估結果......................29
陸、結論................................................31
柒、參考文獻............................................32
捌、圖表................................................37
玖、附錄................................................51
作者簡介................................................55

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