臺灣博碩士論文加值系統

為生產高品質溫帶梨，臺灣每年冬季自日本及中國大陸進口帶有花芽之溫帶梨枝條作為接穗，嫁接於中低海拔橫山梨之徒長枝。蘋果黃化葉斑病毒 (Apple chlorotic leaf spot virus, ACLSV) 為國外普遍感染薔薇科果樹且造成經濟危害之重要植物病毒，因此被列為梨接穗輸入時須檢測之病原種類，現行 ACLSV 之檢測主要以酵素連結免疫吸附分析 (enzyme-linked immunosorbent assay, ELISA) 及反轉錄聚合酶連鎖反應 (reverse transcription polymerase chain reaction, RT-PCR) 為主，然此等方法需要特別儀器，且獲得偵測結果所需之時間仍長，有時亦會有偽陽性之問題產生。反轉錄恆溫環狀擴增法 (reverse transcription loop-mediated isothermal amplification, RT-LAMP) 是一種可在恆溫下反應且即時、簡便具高專一性及高效率之核酸增幅方法，可直接以肉眼辨識反應結果。為改善現行檢測效率，擬研發以 RT-LAMP 來檢測 ACLSV，首先合成 ACLSV不同病毒分離株之鞘蛋白基因 (coat protein gene, CP) DNA 為標的序列，並利用胞外轉錄合成 RNA 做為本試驗之正對照組病毒核酸，同時針對各鞘蛋白基因區域設計共4條之專一性引子組，並於恆溫63˚C下進行60分鐘增幅反應可順利偵測到 ACLSV，但對其他亦普遍感染梨樹之病毒如蘋果莖痘斑病毒 (Apple stem pitting virus) 及蘋果莖凹陷病毒 (Apple stem grooving virus) 則無法被增幅。為比較 RT-LAMP 與 RT-PCR 兩者之靈敏度，將 ACLSV 之 A4 分離株 RNA 經過10倍序列稀釋並混合健康梨 RNA 後，同時進行此兩反應，發現 RT-LAMP 於增幅70分鐘時其靈敏度較 RT-PCR 高出100倍。而比較 RT-LAMP 引子組對三分離株增幅之靈敏度差異，發現 A4 分離株增幅之靈敏度較 Kuerle 及 YT-2-CS 分離株高，可歸因於引子組對於 A4 分離株有較佳之黏合性。接著實際以所建立之 RT-LAMP 技術對2013至2014年輸入的其中473枝梨接穗樣本進行篩檢，並輔以 ELISA 及 RT-PCR 技術做重複確認，發現得到一致結果。綜合以上研究，RT-LAMP 較現行方法縮短檢測時程，提高檢測效率，兼具專一及靈敏的特性，期能藉以對輸入梨接穗之 ACLSV 提供更快速、方便、準確且靈敏之檢測方式。

To produce high quality pear, branches with flower buds of pear grown in temperate region are imported from Japan and China to serves as scions to suckers of Hengshan pear in Taiwan. However, an important plant virus, Apple chlorotic leaf spot virus (ACLSV), commonly infects Rosaceae fruit trees and causes economic damages in foreign countries. Therefore, ACLSV has been listed as a quarantine pathogen. Currently, the detection of ACLSV is mainly conducted by use of enzyme-linked immunesorbent assay (ELISA) or reverse transcription polymerase chain reaction (RT-PCR). However, both ELISA and RT-PCR require specific equipments and are also time-consuming. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a real-time, simple and highly efficient method to amplify nucleic acid and can be processed under an isothermal condition. Furthermore, the result of RT-LAMP can be observed by change of colors without a specific equipment. To improve current detection methods, this research plans to develop RT-LAMP for the detection of ACLSV. We synthesized DNA of the coat protein (CP) genes derived from three isolates of ACLSV, and use in vitro transcription to generate ACLSV RNA as the positive control. We have optimized the RT-LAMP detection, and the amplification of ACLSV can be achieved at 63˚C for 60 minutes using a set of 4 specific primers targeting the viral CP genes. In addition, the RT-LAMP can only detect ACLSV but not viruses commonly infect pear such as Apple stem pitting virus and Apple stem grooving virus. To compare the sensitivity of RT-LAMP with that of RT-PCR, RNA isolated from A4 isolates of ACLSV was diluted serially in 10-fold increments and added to RNA from healthy pear for the assays. After 70 minutes of RT-LAMP reaction, the result showed that the detection sensitivity of the RT-LAMP was 100 times higher than that of the RT-PCR. Besides, comparing the detection sensitivity of the RT-LAMP primers to the three isolates of ACLSV, the result showed that A4 isolate was higher than that of Kuerle and YT-2-CS isolates, which was attributed to better combination ability of the primers to the sequence of A4 isolate. A total of 473 pear scions imported from 2013 to 2014 were screened by the developed RT-LAMP and confirmed by ELISA and RT-PCR. The results of ACLSV detection by use of the three methods were consistent. It suggests that the developed RT-LAMP is convenient and time-saving without sacrificing specificity and sensitivity.

誌謝 I
中文摘要 II
Abstract IV
目錄 VI
表目錄 IX
圖目錄 X
壹、前言 1
貳、前人文獻探討 3
一、梨樹栽培概況 3
二、臺灣梨接穗檢疫法規及輸入概況 4
(一) 臺灣梨接穗檢疫法規沿革 4
(二) 臺灣梨接穗近年輸入概況 6
三、感染梨樹重要病毒種類及其特性 6
(一) 蘋果黃化葉斑病毒 (Apple chlorotic leaf spot virus, ACLSV) 6
(二) 蘋果莖痘斑病毒 (Apple stem pitting virus, ASPV) 8
(三) 蘋果莖凹陷病毒 (Apple stem grooving virus, ASGV) 9
(四) 蘋果嵌紋病毒 (Apple mosaic virus, ApMV) 10
(五) 梨潛隱病毒 (Peach latent mosaic viroid, PLMVd) 10
(六) 番茄叢生矮化病毒 (Tomato bushy stunt virus, TBSV) 10
四、蘋果黃化葉斑病毒 (ACLSV) 之風險潛能 (Pest risk potential) 11
(一) 傳入之後果評估 11
(二) 傳入可能性之風險評等 12
(三) 蘋果黃化葉斑病毒 (ACLSV) 之風險潛能評估結果 13
五、現行輸入梨接穗常用病毒檢測技術 14
(一) 血清學檢測技術 14
(二) 核酸增幅技術 14
(三) 現行檢測技術待改善之處及研究動機 14
六、恆溫環狀擴增法之原理及特性 15
七、應用恆溫環狀擴增法檢測植物病原之實例 16
(一) 病毒與類病毒 16
(二) 真菌 17
(三) 細菌 18
(四) 植物菌質體 18
(五) 線蟲 19
參、材料與方法 20
一、供試植物檢測材料 20
二、供試目標病毒製備 20
(一) 篩選欲合成之 ACLSV 分離株 20
(二) 合成病毒鞘蛋白核酸序列 21
(三) 擴增所合成病毒鞘蛋白核酸序列及保存 21
(四) 抽取質體 DNA 及確認目標基因 21
(五) 限制酶作用及核酸純化 22
(六) 生體外轉錄 (In vitro Transcription) 及 RNA 轉錄本之純化與保存 23
三、RT-LAMP 專一性引子組之設計 24
四、引子組檢測之反應條件及環境測試 24
五、混合病毒與植物核酸同時進行 ACLSV 及梨之 actin gene檢測 25
六、評估 RT-LAMP 檢測 ACLSV 病毒之專一性 25
七、評估 RT-LAMP 偵測 ACLSV 病毒之靈敏度 26
八、對輸入梨接穗樣本進行病毒檢測 26
(一) 植物全 RNA 之純化 26
(二) 反轉錄恆溫環狀擴增法 (RT-LAMP) 27
(三) 反轉錄聚合酶連鎖反應 (RT-PCR) 28
(四) ELISA 檢測 29
肆、結果 31
一、目標病毒序列分析以篩選欲合成之 ACLSV 分離株 31
二、抽取質體 DNA 後確認轉型成功 31
三、以限制酶作用確認 ACLSV 之鞘蛋白基因選殖成功 32
四、供試病毒之 RNA 轉錄本 (transcript) 製備 32
五、檢測 ACLSV 之 RT-LAMP 引子組設計 32
六、檢測 ACLSV 引子組之反應條件及環境測試 32
七、檢測梨肌動蛋白基因 (actin gene) 之 RT-LAMP 引子組………………..33
八、混合病毒與植物核酸同時進行 ACLSV 及梨之 actin gene 檢測………33
九、評估 RT-LAMP 檢測 ACLSV 之專一性 34
十、評估 RT-LAMP 檢測 ACLSV 之靈敏度 34
十一、以RT-PCR 檢測 ACLSV 35
十二、以 RT-PCR 檢測梨肌動蛋白基因 (actin gene) 35
十三、輸入梨接穗檢疫時採樣之樣本進行病毒檢測 35
伍、討論 38
陸、結論 81
柒、參考文獻 82

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