Multiplex diagnostic for biothreat C. psittaci & non-threat respiratory pathogens

描述(由申请人提供)：迫切需要为NIAID A、B和C类生物治疗病原体开发快速样本到答案的诊所和现场可部署的诊断方法，以保护军事和平民人口。这项应用的目标是推进和验证一种主要的候选诊断方法-NetBio最近开发的一种新型微流体核酸检测和测序诊断平台-以识别B类病原体鹦鹉热衣原体(CPS)，该病原体可导致危及生命的呼吸系统疾病，历史上一直是生物武器开发的焦点，也是一种研究很少的病原体。此外，我们亦会发现导致非典型肺炎的主要病原体和非生物疗法病原体：沙眼衣原体(Ct)、肺炎衣原体(Cpn)、肺炎支原体(MP)和嗜肺军团菌(Lp)。事实上，我们对生物防御RFA-AI-11-014做出回应的状态是，“使用平台快速区分个人感染了生物威胁因子还是具有类似的全身症状的普通感染的医疗诊断是高度优先的。”目前，美国很少或根本没有针对这些病原体的商业诊断方法。NetBio平台由一个坚固耐用的分析仪器组成，该仪器接受生物芯片组(BCS)--如初步研究中所示，用于从临床样本中提纯基因组DNA、扩增、电泳法分离以及激光检测和Ct型。整个系统从DNA提纯到检测大约需要1个小时。其目的是：1)具有代表性的CPS非典型呼吸道病原体基因组序列，用于可靠的引物筛选；2)改进BCS，从人类临床鼻咽拭子和痰标本中提纯基因组DNA；3)利用Dr.Read开发的引物选择流水线，根据现有基因组和目标1中测序的比较基因组学鉴定用于区分生物和非生物硫酸盐非典型呼吸道病原体的引物，并建立用于DNA扩增的多重DNA扩增方法，以区分每种病原体；4)与现有的Ct、Cpn、MP和Lp的商业核酸扩增试验(Cp不存在)进行比较，并与FDA批准的临床试验之前的高灵敏度的内部RT-PCR试验进行比较，评估NetBio检测方法的敏感性、特异性以及阳性和阴性预测价值。未来的目标是将该检测扩大到其他生物治疗呼吸道病原体。我们预计NetBio样本到答案测试将在急诊室、MD办公室、诊所、军事设施、医院和外地使用，以促进我们的理解 了解非典型呼吸道疾病的流行病学和最佳治疗战略，这将为生物治疗的准备工作提供信息。广泛使用的诊断方法将检测患有常见但也有生物感染的患者，从而能够及早识别攻击并快速治疗受感染的军人和平民人口。

DESCRIPTION (provided by applicant): There is an urgent need to develop rapid sample-to-answer clinic and field deployable diagnostics for NIAID Category A, B and C biothreat pathogens to protect both military and civilian populations. The goal of this application is to advance and validate a lead candidate diagnostic - a novel microfluidics nucleic acid detection and sequencing diagnostic platform recently developed by NetBio-to identify Category B pathogen Chlamydia psittaci (Cps) that causes life-threatening respiratory diseases and has historically been a focus of bioweapons development as well as being a vastly understudied pathogen. We will also detect emerging Cps pathogens and non-biothreat pathogens that are the leading cause of atypical pneumonia and often confused with biothreat agents in clinical presentation: Chlamydia trachomatis (Ct), Chlamydia pneumoniae (Cpn), Mycoplasma pneumoniae (Mp) and Legionella pneumophila (Lp). Indeed, the Biodefense RFA-AI-11-014 we are responsive to states, "Medical diagnostics that use platforms to rapidly distinguish whether an individual is infected with a biological threat agent or a common infection with similar, generalized symptoms are of high priority." There are currently few or no commercial diagnostics for these pathogens in the US. The NetBio platform consists of a ruggedized, analytic instrument that accepts a biochipset (BCS)-shown in Preliminary Studies to purify genomic DNA from clinical samples, amplify, electrophoretically separate and laser detect and type Ct. The entire system from DNA purification to detection takes ~1 hour. The Aims are to: 1) genome sequence representative Cps biothreat and non- biothreat atypical respiratory pathogens for robust primer selection; 2) modify the BCS to purify genomic DNA from human clinical nasopharyngeal (NP) swab and sputum samples; 3) employ the primer selection pipeline developed by Dr. Read to identify primers based on comparative genomics of available genomes and those sequenced in Aim 1 for differentiating biothreat and non-biothreat atypical respiratory pathogens, and develop a multiplexed assay for DNA amplification to distinguish each pathogen; and 4) evaluate the sensitivity, specificity and positive and negative predictive value of the NetBio assay using first spiked samples and then clinical NP swabs and sputum samples compared to the available commercial nucleic acid amplification tests for Ct, Cpn, Mp and Lp (none exist for Cps), and compared to highly sensitive in-house RT-PCR assays prior to clinical trials for FDA approval. A future goal will be to expand the assay to other biothreat respiratory pathogens. We envision that the NetBio sample-to-answer assay will be used in ERs, MD offices, clinics, military facilities, hospitals and the field to advance our understanding of the epidemiology of atypical respiratory diseases and best treatment strategies, which will inform biothreat preparedness. A broadly used diagnostic will detect patients with common but also biothreat infection, enabling early identification of an attack and rapid treatment of infecte military and civilian populations.