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題 名 | A DNA Probe for Identification of Xanthomonas Campestris pv. Campestris, the Causal Organism of Black Rot of Crucifers in Taiwan=鑑定臺灣十字花科蔬菜黑腐病菌之核酸探針 |
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作 者 | 石信德; 林元春; 黃秀珍; 曾國欽; 徐世典; | 書刊名 | Botanical Bulletin of Academia Sinica |
卷 期 | 41:2 2000.04[民89.04] |
頁 次 | 頁113-120 |
分類號 | 435.2 |
關鍵詞 | 十字花科蔬菜; 黑腐病菌; 核酸探針; 偵測; 鑑定; Crucifers; Brassica spp; Detection; DNA probe; Identification; Xanthomonas campestris pv campestris; |
語 文 | 英文(English) |
中文摘要 | 十字花科蔬菜黑腐病菌 (Xanthomonas campestris pv. campestris) Xcc70 菌林之全 DNA 經 EcoRI 切割後,選殖於質體 pBluescript II KS 內,再轉形至大腸桿菌 DHl0B 中。由此隨機選取之選殖株,將其重組質體以 digoxigenin 標識後作為探針,用於篩選其對鑑定黑腐病菌臺灣菌株之專一性。在南方雜配分析上,其中一個選殖株 (pXcc70-8) 僅能與黑腐病菌全 DNA 以 EcoRI 切割後之數個片段產生雜配訊號。此選殖株內之黑腐病菌嵌入 DNA 片段以 EcoRI 切割後含有 2.7,1.6 及 0.6 kb 三個片段,將其中之 0.6 kb 片段藉由次選殖後所製備的探針 (Xcc70-8-1) 與黑腐病菌所有 51 個菌株及蔬菜細菌性葉斑病菌 (X. campestris pv. armoraciae) 7 個菌株均有雜配訊號,而與其他細菌之 60 個供試菌株,則無任何雜配反應。當黑腐病菌及細菌性葉斑病菌的全 DNA 以 KpnI 切割後,此探針在所測試的 51 個黑腐病菌株,均可雜合出單一條 0.7 kb 片段,而在所測試的 7 個葉斑病菌株,則可雜合出單一條 2.5 kb 片段。此探針 Xcc70-8-1 能偵測黑腐病菌 DNA 量及細胞數之最低限度分別為 25 pg 及約 6 x l04 CFU。利用菌落及點漬雜配法偵測罹病甘藍葉片及數種十字花科蔬菜種子磨碎液中的黑腐病菌,結果顯示此 DNA 探針可用於偵測植物組織內的黑腐病菌,但較不適用於種子之偵測。在臺灣黑腐病流行病學研究上,此探針可作為快速及正確鑑定黑腐病菌之有用工具。 |
英文摘要 | A DNA probe was developed for identification of strains of Xanthomonas campestris pv. campestris from Taiwan. EcoRI restriction fragments of total DNA from X. campestris pv. campestris strain Xcc70 were cloned into pBluescript II KS and transformed into Escherichia coli DH10B. The recombinant plasmid DNAs from clones randomly selected were labeled with digoxigenin and screened for their specificity for X. campestris pv. campestris. In Southern hybridization, one of the clones (pXcc70-8) hybridized to several EcoRI-digested fragments of total DNA from only strains of X. campestris pv. campestris. Digestion of the insert DNA of the clone pXcc70-8 with EcoRI yielded fragments of 2.7, 1.6 and 0.6 kb. When a subclone containing the 0.6 kb fragment was used as a probe (Xcc70-8-l), it hybridized with all 51 strains of X. campestris pv. campestris and all seven strains of X. campestris pv.armoraciae, but not with the 60 strains of other bacteria tested. This probe, however, distinguishably detected a single fragment of 0.7 kb in strains of X. campestris pv. campestris and a single fragment of 2.5 kb in strains of X. campestris pv. armoraciae when their total DNAs were digested with KpnI. The detection limits of the probe Xcc70-8-l for the amount of DNA was 25 pg and for the number of cells was about 6 ?104 CFU in dot blot assays. The colony and dot blot hybridizations with the probe were used to detect X. campestris pv.campestris in extracts of infected leaves of cabbage and seeds of several crucifers. The results indicate that the DNA probe can be used to detect X. campestris pv. campestris in plant tissues but probably not in seeds. The probe could be a useful tool for rapid identification of the pathogen in epidemiological studies in Taiwan. |
本系統中英文摘要資訊取自各篇刊載內容。