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

描述（由申请人提供）：将开发导致败血症和社区获得性肺炎（CAP）的病原体的高级诊断分析，在临床环境中进行测试，并进入制造原型。检测将基于实时PCR和TaqMan(r)探针-引物组合来检测病原体基因组。实时PCR被认为是低水平核酸检测的金标准，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6个数量级的定量数据。这种高灵敏度，判别和定量可以完成，即使有一个压倒性的背景高度相关的核酸。我们在生物防御方面取得的特征发现和检测设计的进展将应用于更常见的临床病原体。Keim实验室（TGen-NAU）、应用生物系统公司（仪器和分析制造）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。工业合作伙伴以试剂、信息学支持、劳动力和仪器的形式投入了大量资源，以实现该项目的成功。

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.