DNA Microchips: Detecting Microbes in Oral Cavity Fluids

描述(申请人提供)：人类口腔含有复杂的微生物组合，与局部和系统疾病的表现或预防有关。此外，口腔是在生物恐怖袭击过程中释放的潜在危及生命的毒剂的主要入境口岸之一。传统的以培养为基础的监测口腔微生物的方法是不可行的，因为它们很耗时，而且只捕获了口腔微生物区系的一小部分。该项目使用基于核酸的杂交分析作为基于培养的方法的替代方法。具体地说，靶向特异和通用的高密度DNA微芯片将用于快速可靠地检测口腔液中的微生物生物标志物。 将使用两种一般格式的DNA微阵列：(I)口腔芯片，由亲核糖体和真核糖体RNA靶向的寡核苷酸探针的分层集合组成；以及(Ii)通用六元芯片，包含所有可能的(4096)DNA六聚体。为了进一步提高其诊断能力，口腔芯片将扩大到包括检测与微生物群落结构和毒力有关的基因表达的探针。将采用创新的统计和数学工具对这两种格式的数据进行综合分析(即人工神经网络与传统统计方法，如集群分析相结合)。这一新的方法有望(I)提供与口腔微生物群落相关的种群和社区特有的微生物区系特征模式，(Ii)产生快速和明确的生物制剂检测，(Iii)确定格式的限制(敏感性、分辨率)，以及iv)以高空间和时间分辨率揭示口腔微生物区系的结构和功能属性，特别是与局部和系统性疾病的表现和预防相关的属性。

DESCRIPTION (provided by applicant): The human oral cavity harbors complex microbial assemblages, which are associated with the manifestation or prevention of local and systemic disease. In addition, the oral cavity is one of the main ports of entry for potentially life-threatening agents released in the course of a bioterroristic attack. Conventional culture-based approaches for monitoring microbes in the oral cavity are not feasible since they are time-consuming and capture only a minor fraction of the oral microbiota. This project uses nucleic acid-based hybridization assays as an alternative to culture-based approaches. Specifically, target-specific and generic high-density DNA microchips will be used for the fast and reliable detection of microbial biomarkers in oral fluids. Two general formats of DNA microarrays will be used: (I) the OralChip, consisting of hierarchical sets of pro-and eukaryal ribosomal RNA-targeted oligonucleotide probes, and (II) the generic HexamerChip, containing all possible (4096) DNA hexamers. To further increase its diagnostic power, the OralChip will be expanded to include probes that detect the expression of genes involved in microbial community structure and virulence. Innovative statistical and mathematical tools will be applied for the integrated data analysis of both formats (i.e. artificial neural networks combined with conventional statistical approaches such as cluster analyses). This novel approach is expected to (i) provide population- and community-specific signature patterns of microbiota associated with oral microbial communities, ii) yield fast and unambiguous detection of biothreat-agents, (iii) determine the limitations (sensitivity, resolution) of the formats, and iv) reveal structural and functional attributes of the oral microbiota at high spatial and temporal resolution, specifically those associated with manifestation and prevention of local and systemic disease.