鑑定及偵測瓜類細菌性果斑病菌之聚合酵素連鎖反應技術

Abstract

本研究利用RAPD (random amplified polymorphic DNA)技術增幅並篩選出Acidovorax avenae subsp. citrulli特有之DNA片段D11-300後，將其選殖出來並分析其核酸序列，再依據其核酸序列設計出對A. avenae subsp. citrulli具專一性之引子對SL1/SR1。利用此專一性引子對進行聚合酵素連鎖反應 (polymerase chain reaction, PCR)對供試之所有西瓜、洋香瓜及苦瓜等不同寄主來源之A. avenae subsp. citrulli皆能增幅出194 bp的片段，而對其他7屬16種細菌則否。當以引子對SL1/SR1測試果斑病菌之染色體DNA時，其靈敏度為100 pg，而用於偵測其細菌數時靈敏度可以達到1.25102 cfu，若進行第二次PCR反應(second PCR)則可達1.2 cfu。西瓜及洋香瓜分離的三種腐生菌在濃度為A. avenae subsp. citrulli的100倍時，不影響引子對SL1/SR1之靈敏度。利用此引子對進行PCR可於四小時內鑑定出培養之瓜類細菌性果斑病菌，當應用於瓜類罹病之葉片及果實之診斷時皆可測得特定的194bp片段，同時也皆可分離到果斑病菌。至於應用PCR技術偵測瓜類種子時，從人工混菌之西瓜、洋香瓜種子及自然帶菌之西瓜種子亦可測得特定的194bp片段，但只有少部份樣品能分離出果斑病菌。由上述各項結果顯示引子對SL1/SR1具有快速鑑定果斑病菌及診斷果斑病之功用，在種子檢測上也有應用潛力。

Sixty different random primers were used to find specific DNA fragments of Acidovorax avenae subsp. citrulli by using random amplified polymorphic DNA (RAPD). A specific DNA fragment (about 300bp in size) of A. avenae subsp. citrulli amplified by the primer OPD-11 was cloned into the pCRII-TOPO vector, and sequenced to design primer pairs SL1/SR1. The primer pairs could amplify a distinct band of 194 bp that was specific to A. avenae subsp. citrulli isolated from watermelon, muskmelon and bitter gourd by polymerase chain reaction (PCR). And there was no any DNA fragment amplified with primer pairs SL1/SR1 from any other tested bacteria belonging to 16 species and 7 genus. The minimum amount of DNA from A. avenae subsp. citrulli that could be amplified by PCR was 100pg. Sensitivity of PCR for detection of cells of A. avenae subsp. citrulli with primer pairs SL1/SR1 was 1.2'102 cfu, and could be raised to 1.2 cfu by re-amplification of diluted standard PCR products (Second PCR). Nontarget bacteria WS, MS1 and MS2 did not affect the efficiency of specific amplification of A. avenae subsp. citrulli in PCR assay with primer pairs SL1/SR1. PCR technique with primer pairs SL1/SR1 identifies cultures of A. avenae subsp. citrulli within 4hours and detected the bacterium in diseased tissues and infested seeds of watermelon and muskmelon. The results indicated that the primer pairs SL1/SR1 could be a useful tool for rapid identification and detection of A. avenae subsp. citrulli in diseased plant tissues infected with A. avenae subsp. citrulli, and could also be potentially used for detection of contaminated seeds.