Multiplex Polymerase Chain Reaction for Simultaneous Detection of Xanthomonas campestris pv. campestris and X. campestris pv. raphani

Abstract

Black rot of crucifers caused by Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc) is an important disease worldwide. In recent years, the bacterial leaf spot of Brassica spp. caused by X. campestris pv. raphani (White) Dye (Xcr) has been reported and has caused serious economic losses in several countries. The primary inoculum sources of these two pathogens of crucifers are mainly from the infected or contaminated seeds or seedlings. For disease diagnosis and quarantine purpose, a specific and rapid detection technique for Xcc and Xcr is needed. In this study, the genomic suppression subtractive hybridization (SSH) method was used to obtain specific DNA fragments for Xcc or Xcr. Six and 9 clones were randomly selected from Xcc-Xcr and Xcr-Xcc SSH library, respectively. The insert DNA of these clones were sequenced and blasted to the NCBI and JCVI database. Two candidates of specific DNA fragments, Xcc 34-2 and XLS 2-14 were selected. According to the sequences of specific DNAs fragments in clones of Xcc 34-2 and XLS 2-14, primer pairs Xcc 2f/2r and Xcr 14f/14r were designed for the detection and identification of Xcc and Xcr. The DNA fragments of 200 bp and 277 bp were specifically amplified from strains of Xcc and Xcr by multiplex PCR with these two primer pairs. The detection sensitivity of primer pairs Xcc 2f/2r and Xcr 14f/14r was 10 pg and 100 pg DNA for Xcc and Xcr, respectively. When the black rot and bacterial spot naturally infected leaf tissues or seeds of crucifers were examined, Xcc and Xcr could be detected and identified specifically and simultaneously by multiplex PCR. The detection technique developed in this study could be used to differentiate the diseases caused by Xcc and Xcr, and it could also be used to detect Xcc and Xcr from cruciferous seeds. Keywords: Xanthomonas campestris pv. Campestris;Xanthomonas campestris pv. Raphani;multiplex PCR Xanthomonas campestris pv. campestris (Xcc) 所引起之黑腐病為十字花科作物重要之病害，近年來 X. camestris pv. raphani (Xcr) 所引起之十字花科細菌性葉斑病已在許多國家發生，並造成嚴重之損失。感染或污染此兩種病原細菌之種子或種苗為其最初感染源，因此開發此兩種植物病原細菌之準確、靈敏且快速之檢測技術，為病害診斷與植物檢疫所亟需。本研究應用基因體減扣法分別篩選出 Xcc 與 Xcr 特異性核酸片段，依其核酸序列設計出 Xcc 與 Xcr 之專一性引子對 Xcc 2f/2r 與 Xcr 14f/14r，測試此兩組引子對之專一性與靈敏度，顯示此兩組引子對可分別對Xcc 與 Xcr 增幅出 200 bp 與 277 bp 之核酸片段，其相對應之靈敏度分別為 10 pg 與 100 pg。利用此兩組引子進行多引子聚合瓷連鎖反應測試，顯示此技術可同時檢測與區別此兩種病原細菌，此技術可應用於十字花科黑腐病與細菌性葉斑病之診斷與帶菌種子之檢測。