- 學位論文
應用反轉錄恆溫環狀擴增法偵測輸入梨接穗之蘋果黃化葉斑病毒
The detection of Apple chlorotic leaf spot virus from imported pear scions by the method of reverse transcription loop-mediated isothermal amplification (RT-LAMP)
摘要
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。
並列摘要
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.