| 論文中文題目 |
火鶴花細菌性葉斑病菌 Acidovorax anthurii 專一性引子開發 |
| 論文英文題目 |
A specific primer pair for diagnosis and detection of bacterial leaf spot pathogen: Acidovorax anthurii on anthurium. |
| 論文中文摘要 |
利 用 隨 機 增 幅 多 型 性 核 酸 技 術 ( r a n d o m a m p l i f i e d polymorphic DNA,RAPD),以 160 個逢機引子增幅火鶴花細菌性葉斑病菌 Acidovorax anthurii 的基因體 DNA,篩選出 A. anthurii 特有的 1813 bp 專一性片段,並進一步將此 DNA 片段純化,選殖後進行轉形質體 pW2 Ant -1813 bp 的核酸序列分析,再根據其序列設計對 A. anthurii 具有專一性的引子組 Ant 2F/Ant 2R。應用此引子組進行聚合酵素連鎖反應 (polymerase chain reaction, PCR),供試的 11 株火鶴花細菌性葉斑病菌的菌株皆能增幅出 343 bp 的片段,但對其他供試的 8 屬 21 種共 121 菌株的其他非標的病原細菌及腐生細菌,均不會產生任何片段。以 Ant 2F/Ant 2R 引子組測試細菌性葉斑病菌的 DNA, 其靈敏度可達 10 ~ 50 pg,菌落偵測的靈敏度為 4.5×101 cfu/ml 活菌數。將 106 cfu/ml 的 A. anthurii 菌液分別與其他細菌的菌液 105 ~ 108 cfu/ml 等比率混合後,再進行聚合酵素連鎖反應, 也不影響 A. anthurii 的偵測結果。應用單一菌落快速檢測法可於 3 - 4 小時內鑑定火鶴花細菌性葉斑病菌,顯示本研究所設計的引子組 Ant 2F/Ant 2R 可用於火鶴花細菌性葉斑病菌快速診斷鑑定及檢測。 |
| 論文英文摘要 |
A specific PCR (polymerase chain reaction) primer pair has been developed to detect Acidovorax anthurii using RAPD (random amplified polymorphic DNA). A 1813 bp DNA fragment was amplified from genomic DNA of A. anthurii using OPW-2 primer, after screening one hundred and sixty random primers in RAPD. The amplified DNA was purified, cloned into a pCR II-TOPO vector, and sequenced. A primer pair Ant 2F/Ant 2R based on 1813-bp DNA sequence was designed and tested for specificity. The primer pair could amplify a DNA fragment of 343 bp that was specific to Acidovorax anthurii. No DNA fragment was amplified by the same primer pair from other 21 bacterial species in 8 genera, including non-target pathogens and saprophytes. The designed primer pair Ant 2F/Ant 2R was highly sensitive and could amplify the 343-bp fragment from A. anthurii DNA between 10-50 pg obtained from 4.5×101 cfu/ml bacterial cells. Non-target bacterial DNA did not affect the efficiency of specific amplification of A. anthurii in PCR assays with the primer pair Ant 2F/Ant 2R. Successful identification of A. anthurii could be achieved within 3-4 hrs using PCR with specific primer set. Based on the data provided, we conclude that the primer pair Ant 2F/Ant 2R will be a useful tool for rapid identification and diagnosis of Acidovorax anthurii. |
| 論文中文關鍵字 |
- 火鶴花
- 細菌性葉斑病
- Acidovorax anthurii
- 引子
- 診斷
- 鑑定
- 偵測
|
| 論文英文關鍵字 |
- anthurium
- Acidovorax anthurii
- PCR
- primer
- diagnosis
- identification
- detection
|