細菌性葉斑病菌Pseudomonas cichorii專一性引子之開發
Other Title
Development of specific PCR primers for identification of Pseudomonas cichorii
Journal
植物病理學會刊
Journal Volume
15
Journal Issue
4
Pages
275-285
Date Issued
2006-12
Date
2006-12
Author(s)
Abstract
以60個逢機引子應用隨機增幅多型性核酸技術(RAPD)增幅並篩選出對細菌性葉斑病菌Pseudomonas cichorii (Swingle) Stapp特有之1,100 bp的專一性片段，進一步將該片段接入Topo載體pCR®II-TOPO得到選殖株，並分析其核酸序列，再根據其序列設計出對Pseudomonas cichorii具專一性之引子對SfL1/SfR2。應用此引子對進行聚台酵素連鎖反應(polymerase chain reaction, PCR)，其對供試向日葵細菌比葉斑病菌皆能增幅出379 bp的片段，但對供試之6屬21種其他非標的之病原菌及雜菌菌株則不會產生任何片段。當以引子對SfL1 / SfR2測試細菌性葉斑病菌P. cichorii之全DNA時，其靈敏度可偵測到5~10 pg，而用於偵測其細菌數之靈敏度則可以測到5.5~9個活菌數。將向日葵細菌性葉斑病菌(P. cichorii) 10 6 cfu/ml菌液與其他細菌菌株之菌液105~105 cfu/ml混合，再以引子對SfL1/ SfR2進行聚合酵素連鎖反應，結果顯示不影響其對P. cichorii之責則效率。應用單一菌落快速檢定法可於3-4 hr內鑑定向日葵細菌性葉斑病菌，該引子對可用於人工接種葉斑病菌之向日葵植株樣品之偵測，因此確認所設計之引子對SfL1/SfR2可用於快速鑑定及診斷細菌性葉斑病菌P. cichorii。
A specific PCR (polymerase chain reaction) primer pair have been developed using RAPD (random amplified polymorphic DNA ) to detect Pseudomonas cichorii. Totally sixty random primers were used to find specific DNA fragments of P. cichorii, and a specific DNA fragment of 1,100 bp amplified by the primer OPX17 was cloned into the pCR®II-TOPO vector and further sequenced to design a specific primer pair SfL1 / SfR2. The primer pair could amplify a distinct band of 379 bp that was specific to P. cichorii, and no DNA fragment amplified by the same primer pair from the other tested 21 bacterial species in 6 genera. Sensitivity of PCR for detection of P. cichorii with primer pair SfL1 / SfR2 was between 5 ~ 10 pg for purified DNA and 5.5 ~9 cfu for cultured cells. Non-target bacteria did not affect the efficiency of specific amplification of P. cichorii in PCR assay with primer pair SfL1 / SfR2. PCR technique using primer pair SfL1 / SfR2 identifies the culture of P. cichorii within 3 - 4 hours and detected the bacterium in artificially inoculated sunflower leaf tissue. Based on the data provided, we conclude that the primer pair SfL1 / SfR2 might be a useful tool for rapid identification and diagnosis of P. cichorii.
Subjects
向日葵
細菌性葉斑病菌
PCR引子
Publisher
中華民國植物病理學會
Type
journal article
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