FilmArray Biothreat Pathogens: An Automated Multiplex PCR Diagnostic System

描述（由申请人提供）： 针对美国的蓄意恐怖主义行为表明，必须使我国具备抵御这些行动的手段。2001年的炭疽信件对人类健康产生了严重影响，导致11人住院，5人死亡，以及因无法迅速诊断而产生的恐惧。为了应对这些攻击，爱达荷州技术公司（ITI）创建了能够检测生物战剂的诊断系统。然而，2007年NIAID生物防御战略计划表明，需要从“一个错误，一个解决方案”的方法，能够应对多种威胁生物体的方法。例如，有必要确定十几种生物战剂，并将其与产生类似威胁生命的重大疾病的常见病原体区分开来。另一个需要是高度适应性的方法，这种方法允许随着新的生物威胁剂的出现而快速修改。ITI正在开发一种名为FilmArray（tm）的新系统来满足这些需求，该系统包括实时聚合酶链反应（PCR）仪器和新型袋中实验室，该系统可在1小时内完成从样品纯化到多重微生物检测和鉴定的所有步骤。将单个人体样本注入袋中，然后插入自动执行所有所需步骤的仪器中。该系统具有高度适应性，因为可以使用不同的多重测定来制造测试条。到目前为止，我们已经开发出一种可以在120孔阵列中识别20种呼吸道病原体的袋。在该提案中，ITI将通过开发一种可识别32种微生物的系统来扩展FilmArray的能力。为此，将追求以下目标。目标1。生产可识别生物战剂和常见病原体的FilmArray生物威胁检测试剂。目标2.通过构建224孔第二阶段PCR阵列增加检测到的微生物数量。目标3.开发用于从鼻拭子和脑脊液（CSF）中自动分离细菌和病毒DNA和RNA的FilmArray方法。目标4。开发软件以促进标本中病原体的自动识别。目标5。在ITI、外部实验室和医院使用人体样本（包括鼻拭子、血液和CSF）对FilmArray生物威胁系统进行检测。相关性（参见说明^：FilmArray将满足公共卫生和医院临床实验室对快速、灵敏、特异、易于使用、适应性强且经济高效的医学诊断的需求，并用于床旁应用。FilmArray对多种病原体的自动化和同时检测将区分受生物威胁因子感染的个体或具有类似全身症状的常见感染。

DESCRIPTION (provided by applicant): Deliberate acts of terrorism against the United States demonstrated the importance of equipping our nation with the means to defend against these actions. The anthrax letters in 2001 had severe impacts on human health, resulting in 11 hospitalizations, 5 deaths, and fear based on inability to rapidly diagnose. In response to these attacks, Idaho Technology Inc (ITI) created diagnostic systems capable of detecting biowarfare agents. However, the 2007 NIAID Strategic Plan for Biodefense indicates the need to migrate from the "one bug, one solution" approach, to methods capable of responding to multiple threat organisms. For example, there is a need to identify more than a dozen biowarfare agents and distinguish them from common pathogens that produce similar life threatening critical illness. Another need is for highly adaptable methods, which allow rapid modification as new biothreat agents emerge. ITI is meeting these needs by developing a new system called the FilmArray(tm), which comprises a real-time polymerase chain reaction (PCR) instrument and novel lab-in-a-pouch that performs all steps from sample purification to multiplex organism detection and identification in under 1 hour. A single human sample is injected into the pouch, then inserted in the instrument which automatically performs all required steps. This system is highly adaptable, as pouches can be manufactured with different multiplex assays. Thus far we have developed a pouch that can identify 20 respiratory pathogens, in a 120-well array. In this proposal, ITI will extend FilmArray's capabilities by developing a system that identifies 32 organisms. To do so the following aims will be pursued. Aim 1. Produce FilmArray biothreat assays that identify both biowarfare agents and common pathogens. Aim 2. Increase the number of organisms detected by building a 224-well 2nd stage PCR array. Aim 3. Develop methods for FilmArray automated isolation of bacterial and viral DMA and RNA from nasal swabs and cerebral spinal fluid (CSF). Aim 4. Develop software to facilitate the automated identification of pathogen(s) within a specimen. Aim 5. Validate the FilmArray biothreat system with human samples, including nasal swabs, blood, and CSF at ITI, external laboratories and hospitals. RELEVANCE (See instructions^: The FilmArray will fill a need for rapid, sensitive, specific, easy to use, adaptable, and cost-effective medical diagnostics for public health and hospital-based clinical laboratories, and point-of-care use. FilmArray's automated and simultaneous detection of multiple pathogens will distinguish individuals infected by a biothreat agent or a common infection with similar, generalized symptoms.