Jaing Crystal, Gardner Shea, McLoughlin Kevin, Mulakken Nisha, Alegria-Hartman Michelle, Banda Phillip, Williams Peter, Gu Pauline, Wagner Mark, Manohar Chitra, Slezak Tom
Chemistry, Materials, Earth and Life Sciences, Lawrence Livermore National Laboratory, Livermore, California, United States of America. jaing2/at/llnl.gov
PLoS One. 2008 May 14;3(5):e2163. doi: 10.1371/journal.pone.0002163.
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 or 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.
新出现的已知和未知病原体对公共卫生构成了严重威胁。为预防和应对疾病爆发，迫切需要用于快速检测和鉴定微生物病原体的平台。现有的检测技术无法提供关于已知或新型生物体的广泛功能信息。作为开发此类系统的第一步，我们制备并测试了一系列高密度功能基因阵列，以检测毒力和抗生素耐药机制的相关元件。我们的第一代阵列针对大肠杆菌K12和CFT073菌株、粪肠球菌和金黄色葡萄球菌的基因。我们确定了用于基因家族检测和区分的最佳探针设计参数。当用与这四种目标菌株具有不同系统发育距离的生物体进行测试时，该阵列检测到了属于同一分类家族的细菌中大多数目标基因家族的直系同源物。结合全基因组扩增，该阵列能够检测到飞克级浓度的纯化DNA，这些DNA要么添加到气溶胶样本背景中，要么来自四种目标生物体中一种或多种的组合。这是关于靶向微生物抗生素耐药性和毒力机制的高密度NimbleGen微阵列系统 的首次报道。通过靶向毒力基因家族以及特定生物威胁病原体特有的基因，这些阵列将提供有关环境样本中未知生物体致病潜力和耐药性概况的重要数据。
