Data for: 3342823

Abstract An oligonucleotide microarray hybridization system to differentiate microbial species was designed and tested. Seven microbial species were studied, including one Bacillus and six Pseudomonas strains. DNA sequences near the 5′ end of 16S rRNA genes were aligned and two contiguous regions of high variability, flanked by highly conserved sequences, were found. The conserved sequences were used to design PCR primers which efficiently amplified these polymorphic regions from all seven species. The amplicon sequences were used to design 88 9mer hybridization probes which were arrayed onto glass slides. Single‐stranded, fluorescence‐tagged PCR products were hybridized to the microarrays at 15°C. The experimental results were compared with the Δ G ° values for all matched and mismatched duplexes possible between the synthetic probes and the 16S target sequences of the seven test species, calculated using a ‘virtual hybridization’ software program. Although the observed hybridization patterns differed significantly from patterns predicted solely on the basis of perfect sequence matches, a unique hybridization fingerprint was obtained for each of the species, including closely related Pseudomonas species, and there was a reasonable correlation between the intensity of observed hybridization signals and the calculated Δ G ° values. The results suggest that both perfect and mismatched pairings can contribute to microbial identification by hybridization fingerprinting.摘要 本研究设计并测试了一种用于区分微生物物种的寡核苷酸微阵列（oligonucleotide microarray）杂交系统。研究共纳入7种微生物，包括1株芽孢杆菌属（Bacillus）菌株与6株假单胞菌属（Pseudomonas）菌株。对16S核糖体RNA（16S rRNA）基因5′端附近的DNA序列进行多序列比对后，发现两段连续的高变区域，其两侧翼为高度保守的序列。利用该保守序列设计聚合酶链式反应（PCR，polymerase chain reaction）引物，可从全部7种微生物中高效扩增出该多态性区域。基于扩增子（amplicon）序列，共设计88条9聚体杂交探针（9mer hybridization probes），并将其点制于玻片之上。将单链荧光标记PCR产物置于15℃条件下与微阵列进行杂交反应。通过"虚拟杂交"（virtual hybridization）软件计算得到合成探针与7种受试物种16S靶序列间所有可能的匹配与错配双链的标准吉布斯自由能变（ΔG°），并将实验结果与该计算值进行比对。尽管观测到的杂交模式与仅基于完美序列匹配所预测的模式存在显著差异，但所有受试物种（包括亲缘关系密切的假单胞菌属物种）均获得了独特的杂交指纹图谱，且观测到的杂交信号强度与计算得到的ΔG°值之间呈现出合理的相关性。本研究结果表明，完美匹配与错配配对均可通过杂交指纹图谱法为微生物物种鉴定提供有效支撑。