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

实时 PCR 检测/脓毒症直接检测/CAP Pa

• 批准号: 7087966
• 负责人: Paul Stephen Keim
• 金额: $ 60.16万
• 依托单位: TRANSLATIONAL GENOMICS RESEARCH INST
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7087966
• 关键词: bacterial DNA; bioinformatics; clinical research; communicable disease diagnosis; cooperative study; diagnosis design /evaluation; diagnostic tests; early diagnosis; human tissue; microarray technology; molecular biology information system; nucleic acid quantitation /detection; pneumonia; polymerase chain reaction; septicemia; single nucleotide polymorphism

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.

描述（由申请人提供）：将开发针对引起脓毒症和社区获得性肺炎（CAP）的病原体的先进诊断检测方法，在临床环境中进行测试，并转移到制造原型中。这些检测将基于实时 PCR 和检测病原体基因组的 TaqMan(r) 探针-引物组合。实时 PCR 被认为是低水平核酸检测的黄金标准，可在几个小时内生成数据，而培养方法则需要两天以上。该技术能够进行单分子检测和单核苷酸区分，同时提供六个数量级的定量数据。即使存在大量高度相关的核酸背景，也可以实现这种高灵敏度、辨别力和定量。 我们的生物防御工作在特征发现和检测设计方面取得的进展将应用于更常见的临床病原体。 Keim 实验室 (TGen-NAU)、Applied Biosystems（仪器和化验制造）和亚利桑那州横幅卫生系统（亚利桑那州实验室科学）的临床专业知识之间提议建立三方合作伙伴关系。为了该项目的成功，工业合作伙伴以试剂、信息学支持、劳动力和仪器等形式投入了大量资源。 利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）。 Applied Biosystems 将使用 Assays-on-Demand(r) 管道将这些检测转化为实时 PCR 检测，以便在 TGen、NAD 和 Banner Health 临床实验室进行验证和确认。将使用大量菌株和临床样本流进行验证，以确保测定的特异性。多项经过验证的测定将被推进到应用生物系统公司的制造工厂，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、毒力和抗生素抗性基因的多种组合进行多达 768 次检测。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疾病表现的定制检测系统。此外，还将开发一个信息学系统，用于在验证过程中处理分析数据，并最终在符合 HIPAA 的环境中处理相关的临床数据。包括血液、痰液等在内的临床样本将使用当前标准实验室培养物和其他方案进行分析，同时进行实时 PCR 检测以进行临床验证研究。每次实时 PCR 检测都会生成定量数据，并一起进行分析，以更好地了解细菌负荷与病原体的对比。