Real-Time PCR Assays for Direct Detection of Sepsis and CAP Pathogens

描述(由申请人提供)：将开发对引起败血症和社区获得性肺炎(CAP)的病原体进行先进的诊断分析，并在临床环境中进行测试，并进入生产原型。这些检测将基于实时聚合酶链式反应和检测病原体基因组的TaqMan(R)探针-引物组合。实时聚合酶链式反应被认为是低水平核酸检测的黄金标准，只需几个小时就能产生数据，而培养方法则需要两天以上。这项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。即使在高度相关的核酸的压倒性背景下，这种高灵敏度、区分度和定量也可以实现。我们的生物防御努力在签名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。凯姆实验室(TGen-NAU)、应用生物系统(仪器和化验制造)和亚利桑那州班纳卫生系统(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。工业合作伙伴为这个项目的成功投入了大量的资源，包括试剂、信息学支持、劳动力和工具。 利用最新的生物信息学方法和迅速扩大的基因组数据库，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-SNPs)。应用生物系统公司将使用Assay-On-Demand(R)管道将这些转换为实时PCR分析，以便在TGen、NAD和班纳健康临床实验室进行验证和确认。针对大量菌株和临床标本流的验证将用于确保检测的特异性。多种经过验证的化验将被推进到应用生物系统制造设施，并将利用低密度阵列微卡技术。低密度微卡阵列具有极大的灵活性，可对病原体、毒力和抗生素耐药性基因的多种组合进行多达768种检测。这种制造灵活性将使我们能够批量生产针对脓毒症、CAP甚至每种疾病的特定临床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化验数据，并最终在符合HIPAA的环境中处理相关的临床数据。临床标本，包括血液、痰等，将使用当前标准的实验室培养和其他方案进行分析，同时进行实时PCR分析，以进行临床验证研究。每次实时聚合酶链式反应分析都会产生定量数据，并一起进行分析，以便更好地了解细菌负荷与病原体的对比。

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.