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論文名稱(外文):Development of Thermostatic Nucleic Acid Amplification Technology for Detecting Acute Hepatopancreatic Necrosis Disease (AHPND)
外文關鍵詞:Acute hepatopancreatic necrosis disease(AHPND)Vibrio parahaemolyticusLoop-mediated isothermal amplification(LAMP)Recombinase polymerase amplification (RPA)Litopenaeus vannamei
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2009 年爆發的蝦類急性肝胰腺壞死症(Acute Hepatopancreatic Necrosis Disease, AHPND),是由副溶血弧菌(Vibrio parahaemolyticus) 帶有相關質體之變異株,分泌毒素所引起,造成養殖白蝦大量死亡, 目前已發展出PCR(Polymerase chain reaction)檢測法可於實驗室進行檢 驗。本研究建立環型恆溫核酸擴增法(Loop-mediated isothermal amplification, LAMP) 以及重組酶聚合酶擴增法(Recombinase polymerase amplification, RPA)檢測AHPND的技術,相較於PCR,以單 一溫度擴增核酸,縮短反應時間、不需複雜的設備,再搭配螢光染劑 或側流試紙判讀結果,取代電泳,可應用於現場操作。LAMP於65℃反 應條件下,使用一組6條專門設計的引子針對質體上毒素A的基因序列 進行擴增。LAMP結果的判讀使用螢光染劑,於手持式紫外燈下觀察螢 光差異;RPA於37℃反應條件下,以兩種方法進行目標序列的擴增, 一種為使用兩條標定過的引子,另一種為加入螢光探針增加專一性, 兩種方法皆於反應後使用側流試紙,試紙上的抗體會辨認引子上及探 針上的標定物,直接呈色判讀結果。結合樣品DNA的簡易萃取法,使 LAMP以及RPA檢測過程可在1-2小時內完成,靈敏度及專一性皆不輸 給PCR。本研究初步於高雄地區採樣養殖白蝦,選取胃部組織抽取DNA, 共檢測出多隻白蝦具有陽性反應,感染AHPND。
The outbreak of acute hepatopancreatic necrosis disease (AHPND) from 2009 has caused mass losses in shrimp farms worldwide. The strain of Vibrio parahaemolyticus which obtain a plasmid and secret toxins has been determined as the infectious agent. PCR methods were published and used to perform diagnosis in laboratory. This study developed methods of Loop-mediated isothermal amplification (LAMP) and Recombinase polymerase amplification (RPA) to detect AHPND and aimed to carry out the diagnosis at the farm side. Compared to PCR, the nucleic acid amplification of LAMP and RPA is performed at a constant temperature which shortens the reaction time and excludes complex equipments. The results are directly color presented by fluorescent dye or lateral flow dipstick which substitute for the gel electrophoresis. The LAMP system for detection of AHPND have six primers targeting toxin A gene (333 bp) in the plasmid under 65 ℃condition. LAMP results were observed through the stain of fluorescent dye and showed the color change emitted by ultraviolet light. RPA have designed two methods for amplification of the target sequence, one is using two labeled primers, the other is added the probe to increase the specificity. After reaction at 37 ℃, both methods followed with lateral flow dipstick which antibody labeled recognize RPA amplicons. Combined with the DNA easy extraction method for template preparation, the time needed to complete the diagnosis is only 1-2 hours. The sensitivity and specificity of LAMP and RPA are better or comparable to PCR in this study. We also examined several white shrimps (Litopenaeus vannamei) from Kaohsiung and found positive signals. It revealed the distribution and consideration of AHPND.
中文摘要 Ⅰ
Abstract Ⅱ
一、 前言 1
1. 緒論 1
2. 急性肝胰腺壞死症(Acute hepatopancreatic necrosis disease) 3
3. 定點照護((Point-of-care) 5
4. 環形恆溫核酸擴增法(Loop-mediated isothermal amplification) 7
5. 重組酶聚合酶擴增法(Recombinase polymerase amplification) 9
二、 實驗架構 13
三、 材料與方法 14
1 實驗材料 14
2 白蝦組織DNA 萃取 16
3 細菌培養與計數(CFU/ml) 16
4 細菌DNA 萃取 17
5 建構帶有AHPND ToxA 基因之人工質體 17
6 質體DNA 萃取 18
7 LAMP 引子設計 19
8 優化LAMP 19
9 LAMP 反應 20
10 RPA 方法一 21
11 RPA 方法二 22
四、 結果 24
1 LAMP 及RPA 引子設計 24
2 LAMP 反應及優化 24
3 RPA 反應 25
4 LAMP、RPA 及PCR 反應靈敏度比較26
5 LAMP、RPA 及PCR 專一性比較 27
6 高雄梓官區養殖白蝦篩檢 27
五、 討論 29
六、 圖&表 36
七、 參考文獻 45
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