Real-Time PCR Assays /Direct Detection of Sepsis /CAP Pa

描述（由申请方提供）：将开发用于导致败血症和社区获得性肺炎（CAP）的病原体的高级诊断检测试剂盒，在临床环境中进行检测，并将其转移到生产原型中。该试验将基于实时PCR和TaqMan（r）探针-引物组合，用于检测病原体基因组。实时PCR被认为是低水平核酸检测的金标准，并在几个小时内生成数据，而培养方法需要两天以上。该技术能够进行单分子检测和单核苷酸识别，同时提供六个数量级的定量数据。这种高灵敏度、区分度和定量即使在存在高度相关的核酸的压倒性背景时也可以实现。 我们在生物防御工作中的特征发现和检测设计方面的进展将应用于更常见的临床病原体。Keim实验室（TGen-NAU）、应用生物系统（仪器和检测制造）和亚利桑那州Banner卫生系统（亚利桑那州实验室科学）的临床专业知识之间提出了三方合作伙伴关系。工业合作伙伴已承诺在试剂，信息学支持，劳动力和仪器的形式为该项目的成功显着的资源。 使用最新的生物信息学方法和快速扩展的基因组数据库，我们将识别大量潜在的诊断基因组特征（例如，单核苷酸多态性-SNP）。 应用生物系统公司将使用Assays-on-Demand（r）管道将这些转化为实时PCR检测，以便在TGen、NAD和Banner Health临床实验室进行验证和确认。将使用针对大量菌株和临床标本流的验证来确保检测试剂盒的特异性。多项经验证的检测试剂盒将提前至Applied Biosystems生产工厂，并将利用低密度阵列微卡技术。低密度微卡阵列具有很大的灵活性，可在病原体、毒力和抗生素耐药基因的多种组合中进行多达768次检测。这种制造灵活性将使我们能够大规模生产针对败血症、CAP甚至每种特定临床疾病表现的定制检测系统。此外，将开发一个信息学系统，用于在验证期间处理测定数据，并最终在符合HIPAA的环境中处理相关临床数据。将使用当前标准实验室培养和其他方案分析临床标本（包括血液、痰液等），同时进行实时PCR检测，以进行临床验证研究。将通过每次实时PCR检测生成定量数据，并一起进行分析，以更好地了解细菌载量与病原体的对比。

DESCRIPTION (provided by applicant): Advanced diagnostic assays for pathogens causing sepsis and community acquired pneumonia (CAP) will be developed, tested in a clinical setting and moved into a manufacturing prototype. The assays will be based upon real-time PCR and TaqMan(r) probe-primer combinations that detect the pathogen genomes. Real-time PCR is considered the gold standard of low level nucleic acid detection and generates data in a few hours, versus more than two days for culturing methods. This technology is capable of single molecule detection and single-nucleotide discrimination, while providing quantitative data across six-orders of magnitude. This high sensitivity, discrimination and quantitation can be accomplished even when there is an overwhelming background of highly related nucleic acids. Advances in signature discovery and assay design from our biodefense efforts will be applied to more common clinical pathogens. A three-way partnership is proposed among the Keim Lab (TGen-NAU), Applied Biosystems (instruments and assay manufacturing), and the clinical expertise of the Arizona Banner Health System (Laboratory Sciences of Arizona). The industrial partners have committed significant resources in the form of reagents, informatics support, labor and instruments towards the success of this project. Using the latest bioinformatics approaches and the rapidly expanding genomic databases, we will identify a large number of potential diagnostic genomic signatures (e.g., single nucleotide polymorphisms-SNPs). Applied Biosystems will use the Assays-on-Demand(r) pipeline to convert these into real-time PCR assays for verification and validation at TGen, NAD and in the Banner Health clinical labs. Validation against large panels of strains and stream of clinical specimens will be used to insure assay specificity. Multiple validated assays will be advanced to the Applied Biosystems manufacturing facility and will utilize the low density array micro-card technology. Low density micro card arrays have great flexibility for up to 768 assays in multiple combinations of pathogens, virulence and antibiotic resistance genes. This manufacturing flexibility will allow us to mass produce customized assay systems targeted at sepsis, CAP or even particular clinical disease presentations of each. In addition, an informatics system will be developed for handling assay data during the validation and eventually for handling associated clinical data in a HIPAA compliant environment. Clinical specimens including blood, sputum, etc. will be analyzed using current standard lab culture and other protocols in parallel to the real-time PCR assays for clinical validation studies. Quantitative data will be generated with every real-time PCR assay and will analyzed together, to better understand bacterial loads in contrast to pathogens.