蝴蝶蘭細菌性葉斑病Burkholderia gladioli專一性引子之開發

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.