On demand blood tests for select agent diagnosis

按需血液测试用于选择药物诊断

• 批准号: 8437128
• 负责人: David Alland
• 金额: $ 2.59万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-07 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8437128
• 关键词: Address; Authorization documentation; Bacteremia; Bacteria; Beauty; Biological Assay; Bioterrorism; Blood; Blood Tests; Body Fluids; Buffers; Categories; Characteristics; Clinical; Clinical Sensitivity; Collaborations; Communicable Diseases; Complex; Coupled; DNA; Detection; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostics Research; Disease; Early treatment; Emergency Situation; Ensure; Goals; Hour; Indium; Infection; Influenza; Laboratories; Licensing; Life; Marketing; Methods; Molecular; Morbidity - disease rate; Nested PCR; New Agents; Patients; Plastics; Polymerase Chain Reaction; Preparation; Process; Protocols documentation; Recording of previous events; Risk; ST14 gene; Sampling; Science; Shipping; Ships; Simulate; Specificity; Specimen; Staging; Staphylococcus aureus; Stream; Symptoms; Syndrome; System; Techniques; Testing; Virulence; Work; base; cost; cost effective; design; flexibility; inhibitor/antagonist; innovation; instrument; mortality; pathogen; point of care; prevent; rapid detection; reagent testing

DESCRIPTION (provided by applicant): Diagnostics comprise a critical component of defense against select agent infections. The virulence of most bacterial select agents requires rapid detection and early treatment to prevent serious morbidity and mortality. Most bacterial bioterrorism agents cause blood stream infections (BSI) as part of their disease syndrome. Blood-based detection offers the promise of early BSI diagnosis. However, rapid BSI detection is particularly challenging. Assays must be extremely sensitive to detect early disease. The sensitivity required is on the order of < 5 cfu of the select agent per ml of blood. Yet, the diagnostic assays must also be rapid, robust and easy to perform, preferably near the point-of-care. Even the perfect select agent test will be of little use if it is not be available when neede in an emergency. Thus, the best select agent assays must be able to be manufactured upon very short notice, or be very inexpensive, easy to stockpile, and have a prolonged shelf life. The very best assays would have all of these characteristics. Cepheid and the Alland laboratory have a history of successful collaborations devoted to developing rapid, sensitive, on-demand tests for infectious disease. We recently discovered an approach that is likely to provide highly-sensitive BSI detection. Our approach automatically extracts bacteria from 1 - 10 ml of blood and then automatically performs very-small- amplicon nested PCR. The entire test can be performed within a disposable plastic cartridge and a highly- flexible instrument platform. As proof of principal, our preliminary studies detected S. aureus in the blood of almost 90% of S. aureus BSI patients. Here, we propose to further develop our approach to create rapid point-of-care assays that will detect select agent BSIs at the early stage when a patient may be symptomatic but still have low-level bacteremia. We will also develop a strategy that permits rapid manufacture and distribution of appropriate select agent assays within weeks of a bioterrorist attack with or without stockpiling of an initial test lot. Our six specific aims are t: 1) Develop highly-sensitive, small amplicon nested-PCR assays for each category A select agent bacterium and for each "unconventional" category B bacterial select agent; 2) Develop multiplex assays for major select agent risks; 3) Adapt each assay to the Cepheid cartridge and the GeneXpert(R) platform; 4) Optimize our 1 ml blood processing protocol and develop a method to isolate select agent DNA from 10 ml of blood using Cepheid sample processing cartridges; 5) Perform analytic tests of both uniplex and multiplex assays on simulated clinical samples containing select agents; and 6) Develop an emergency plan for "on demand" manufacture and emergency FDA use authorization.

描述（由申请人提供）：诊断包括防御选择代理感染的关键组件。大多数细菌选择剂的毒力需要快速检测和早期治疗，以防止严重的发病率和死亡率。大多数细菌性生物恐怖剂引起血流感染（BSI）作为其疾病综合征的一部分。基于血液的检测提供了早期BSI诊断的希望。然而，快速BSI检测尤其具有挑战性。检测必须非常敏感，以发现早期疾病。所需的灵敏度为每ml血液<5cfu的选择试剂的数量级。然而，诊断测定还必须是快速、稳健和易于执行的，优选地在护理点附近。如果在紧急情况下无法使用，即使是完美的选择代理测试也将毫无用处。因此，最好的选择试剂测定必须能够在非常短的时间内制造，或者非常便宜，易于储存，并且具有延长的保质期。最好的检测试剂盒应具备所有这些特征。Cepheid和Alland实验室在致力于开发快速、灵敏、按需的传染病检测方面有着成功的合作历史。我们最近发现了一种可能提供高灵敏度BSI检测的方法。我们的方法自动从1 - 10 ml血液中提取细菌，然后自动进行非常小的扩增子巢式PCR。整个测试可以在一次性塑料盒和高度灵活的仪器平台内进行。作为原理的证明，我们的初步研究检测到了S。金黄色葡萄球菌在血液中几乎90%。金黄色葡萄球菌BSI患者。在这里，我们建议进一步开发我们的方法，以创建快速的即时检测方法，该方法将在早期阶段检测选择的药剂BSI，此时患者可能有症状，但仍有低水平的菌血症。我们还将制定一项战略，允许在生物恐怖袭击发生后数周内快速制造和分发适当的选择试剂检测试剂盒，无论是否储存初始检测批次。我们的六个具体目标是：1）为每种A类选择剂细菌和每种“非常规”B类细菌选择剂开发高灵敏度、小扩增子巢式PCR检测; 2）为主要选择剂风险开发多重检测; 3）使每种检测适应Cepheid检测盒和GeneXpert（R）平台; 4）优化我们的1 ml血液处理方案，并开发使用Cepheid样品处理盒从10 ml血液中分离选择性试剂DNA的方法; 5）对含有选择性试剂的模拟临床样品进行单重和多重测定的分析测试;和6）为“按需”生产和紧急FDA使用授权制定应急计划。