蝴蝶蘭細菌性葉斑病Burkholderia gladioli專一性引子之開發
Other Title
Asecific primer pair for diagnosis and detection of bacterial leaf spot pathogen Burkholderia gladioli of Phalaenopsis orchid
Journal
植物病理學會刊
Journal Volume
20
Journal Issue
1-2
Pages
39-51
Date Issued
2011-06
Date
2011-06
Author(s)
Abstract
蝴蝶蘭 細菌性葉斑病是由Burkholderia gladioli (BG)所引起的細菌性病害，本研究以100個逢機引子應用隨增幅 型性核酸技術 (RAPD)增幅，並篩選 BG 共有的 650 bp之專一性片段，並根據其序列設計出對組 BGRPF3/BGRPR3。應用此引子組進行聚合酵素連鎖反應 (polymerase chain reaction, PCR)偵測 BG 菌株皆能 菌株皆能 菌株皆能 增幅出 350 bp 的片段，但對供試之8 屬 21 種共 97 株其他非標的之病原細菌及腐生則不會產任何片段。BGRPF3/BGRPR3引子 組偵測 BG 菌株的DNA 靈敏度可達10 pg，菌落偵測之靈敏度則為1.87 ×10 1cfu/ml個活菌 數。將 BG 菌株與其他非標的細混合液經簡易萃取後進行 PCR，只產生BG 特有的 350 bp片段。應用單一菌 片段。落快速檢定法可於 3–4小時內鑑定蝴蝶蘭細菌性葉斑病。用於偵測人工接種BG 病菌之蝴蝶蘭葉片以及田間自然發病之蝴蝶蘭葉片均具準確性，顯示本研究所設計引子 發病之蝴蝶蘭葉片均具準確性，顯示本研究所設計引子組 BGRPF3/BGRPR3可用於快速鑑定蝴蝶蘭細菌性葉斑病菌。
Bacterial leaf spot disease, caused by Burkholderia gladioli (BG), is a new bacterial disease on the Phalaenopsis cultivation in Taiwan. In this study, a highly specific primer pair was developed for diagnosis and detection of the pathogen. Totally one hundred random primers were used to find specific DNA fragments of BG, and a specific DNA fragment of 650 bp amplified by the primer OPT-20 was identified and cloned into the pCR®II-TOPO vector. The specific DNA fragment of 650 bp was sequenced, and the specific primer pair BGRPF3/BGRPR3 was designed from the sequences for BG identification and detection. In order to elucidate the specificity of the primer pair, totally 97 non-BG lacterial isolates, representing eight genera and twenty-one species, were used for further testing. The results showed that only BG could produce a unique 350 bp fragment. Detection sensitivity of BG using PCR was between 10 pg for purified DNA and 1.87×10 1 cfu/ml for cultured cells, and the sensitivity and specificity were not affected when BG cells were mixed with other non-target bacteria. The detection time for identifying BG cells by PCR with BGRPF3/BGRPR3 primers took noly 3~4 hours. The primer pair was further used for detection BG on infested Phalaenopss orchids and proved to be equally effective.
Subjects
蝴蝶蘭
細菌性葉斑病細菌
引子
診斷
鑑定
偵測
Phalaenopsis
Burkholderia gladioli
primer
diagnosis
identification
detection
Publisher
中華民國植物病理學會
Type
journal article
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