Detection of virulence and antibiotic resistance gene families

Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known and novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples. Keywords: detection, pathogen, virulence mechanism In this report, we describe the process used to design our first generation functional array for highly sensitive detection of virulence and antibiotic resistance gene families. We discuss the probe design algorithms, including virulence gene sequence selection, and our protocols for sample preparation, amplification, labeling, hybridization, and data analysis. We present the results from experiments designed to assess whether the array can detect virulence gene orthologs from organisms without perfect match probes on the array, using both targeted mismatch probes and hybridizations to DNA from other organisms. Also, we report the results from limit of detection studies, using known amounts of bacterial DNA spiked into aerosol samples to measure the minimal concentration required for detection of virulence elements against a complex background.

新发已知与未知病原体对公共卫生构成严峻威胁。亟需构建可快速检测并表征微生物病原体的技术平台，以预防和应对疾病暴发。现有检测技术无法为已知及新型生物体提供全面的功能信息。

为推进此类平台的开发，我们制备并测试了一系列高密度功能基因芯片（high-density functional gene arrays），用于识别毒力与抗生素耐药机制相关元件。本研究的第一代芯片以大肠杆菌（Escherichia coli）K12、CFT073菌株，粪肠球菌（Enterococcus faecalis）及金黄色葡萄球菌（Staphylococcus aureus）的基因为靶向目标。我们优化了用于基因家族检测与区分的探针设计参数。当用与四种靶标菌株系统发育距离各异的生物体进行测试时，该芯片可检测到分类学上属于同一科的细菌中存在的大多数靶标基因家族的直系同源基因（orthologs）。结合全基因组扩增（whole-genome amplification）技术，该芯片可检测到纯化DNA的飞克级浓度，无论是掺入气溶胶样本背景中，还是来自四种靶标生物体中一种或多种的混合DNA样本。

本研究首次报道了一款针对微生物抗生素耐药与毒力机制的高密度NimbleGen微阵列系统。通过靶向毒力基因家族以及特定生物威胁病原体的特异性基因，此类芯片可为环境样本中未知生物体的致病潜力与耐药谱提供关键数据。

关键词：检测、病原体、毒力机制

本报告详述了用于构建第一代功能芯片的流程，该芯片可高灵敏度检测毒力与抗生素耐药基因家族。我们探讨了探针设计算法，包括毒力基因序列筛选流程，以及样本制备、扩增、标记、杂交与数据分析的实验方案。我们展示了相关实验结果：通过设计靶向错配探针，并将芯片与其他生物体的DNA进行杂交，评估该芯片能否检测到阵列上无完美匹配探针的生物体的毒力基因直系同源基因。此外，我们还报道了检测限（limit of detection）研究的结果：通过将已知剂量的细菌DNA掺入气溶胶样本中，测定在复杂背景下检测毒力元件所需的最低浓度。