火鶴花細菌性葉斑病菌Acidovorax anthurii專一性引子開發

A specific primer pair for diagnosis and detection of bacterial leaf spot pathogen: Acidovorax anthurii on anthurium

許秀惠(Hseu, S. H.) ； 蔡宜容(Tsai, Y. J.) ； 曾怡蓉(Tseng Y. J.)

《植物醫學》 58卷1期 (2016/03) Pp. 9-16

https://doi.org/10.6716/JPM.201603_58(1).0002

摘要

利用隨機增幅多型性核酸技術（random amplified 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4.5×10^1 cfu/ml活菌數。將10^6 cfu/ml的A. anthurii菌液分別與其他細菌的菌液10^5～10^8 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。

關鍵字

火鶴花 ； 細菌性葉斑病 ； Acidovorax anthurii ； 引子 ； 診斷 ； 鑑定 ； 偵測

並列摘要

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.

並列關鍵字

anthurium ； Acidovorax anthurii ； PCR ； primer ； diagnosis ； identification ； detection