Phytopathology. 2003 Mar;93(3):262-9. doi: 10.1094/PHYTO.2003.93.3.262.
ABSTRACT Oligonucleotides, 16 to 24 bases long, were selected from the 3' end of the 16S gene and the 16S-23S intergenic spacer regions of bacteria pathogenic on potato, including Clavibacter michiganensis subsp. sepedonicus, Ralstonia solanacearum, and the pectolytic erwinias, including Erwinia carotovora subsp. atroseptica and carotovora and E. chrysanthemi. Oligonucleotides were designed and formatted into an array by pin spotting on nylon membranes. Genomic DNA from bacterial cultures was amplified by polymerase chain reaction using conserved ribosomal primers and labeled simultaneously with digoxigenin-dUTP. Hybridization of amplicons to the array and subsequent serological detection of digoxigenin label revealed different hybridization patterns that were distinct for each species and subspecies tested. Hybridization of amplicons generally was restricted to appropriate homologous oligonucleotides and cross-hybridization with heterologous oligonucleotides was rare. Hybridization patterns were recorded as separate gray values for each hybridized spot and revealed a consistent pattern for multiple strains of each species or subspecies isolated from diverse geographical regions. In preliminary tests, bacteria could be correctly identified and detected by hybridizing to the array amplicons from mixed cultures and inoculated potato tissue.
摘要 从马铃薯致病菌（包括密执安棒杆菌亚种、茄青枯假单胞菌和果胶分解欧文氏菌，包括胡萝卜软腐欧文氏菌亚种和胡萝卜软腐欧文氏菌以及菊欧文氏菌）的 16S 基因和 16S-23S 基因间隔区的 3' 端选择了 16 至 24 个碱基长的寡核苷酸。寡核苷酸通过针点印迹固定在尼龙膜上，并设计和格式化在阵列中。使用保守的核糖体引物通过聚合酶链反应扩增细菌培养物的基因组 DNA，并同时用地高辛-dUTP 标记。将扩增子与阵列杂交，随后对地高辛标记进行血清学检测，揭示了每种测试的物种和亚种的不同杂交模式。扩增子的杂交通常仅限于适当的同源寡核苷酸，异源寡核苷酸的交叉杂交很少。将杂交模式记录为每个杂交斑点的单独灰度值，并揭示了来自不同地理区域的多种菌株的一致模式。在初步测试中，通过与混合培养物和接种的马铃薯组织的阵列扩增子杂交，可以正确识别和检测细菌。
