DNA Microchips: Detecting Microbes in Oral Cavity Fluids

描述（由申请人提供）：人体口腔内含有复杂的微生物聚集体，与局部和全身疾病的表现或预防相关。此外，口腔是生物恐怖袭击过程中释放的可能危及生命的制剂的主要入口之一。用于监测口腔中微生物的常规的基于培养的方法是不可行的，因为它们是耗时的并且仅捕获口腔微生物群的一小部分。该项目使用基于核酸的杂交测定作为基于培养的方法的替代方法。具体而言，目标特异性和通用高密度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。 将使用两种通用形式的DNA微阵列：（I）OralChip，由原核和真核核糖体RNA靶向寡核苷酸探针的分层组组成，和（II）通用HexamerChip，包含所有可能的（4096）DNA六聚体。为了进一步提高其诊断能力，OralChip将扩展到包括检测微生物群落结构和毒力相关基因表达的探针。创新的统计和数学工具将用于两种格式的综合数据分析（即人工神经网络与聚类分析等传统统计方法相结合）。这种新的方法预计（i）提供与口腔微生物群落相关的微生物群的群体和群落特异性特征模式，ii）产生生物威胁剂的快速和明确的检测，（iii）确定生物威胁剂的局限性。（灵敏度，分辨率），以及iv）以高空间和时间分辨率揭示口腔微生物群的结构和功能属性，特别是与局部和全身疾病表现和预防相关的那些。

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.