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(上? @)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合酵素連鎖反應(polymerase chain reaction，PCR)，其對供試向日葵細菌性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5~10pg，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)10^6cfu/ml菌液與其他細菌菌株之菌液10^5~10^8cfu/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之偵測效率。應用單一菌落快速檢定法可於3~4hr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(superscript @)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.
