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类生物威胁病原体开发快速的样本到答案的临床和现场可部署诊断方法，以保护军事和平民。本申请的目标是推进和验证一种主要的候选诊断方法——一种由netbio最近开发的新型微流体核酸检测和测序诊断平台，用于识别B类病原体鹦鹉热衣原体（Cps），这种病原体会导致危及生命的呼吸系统疾病，历来是生物武器开发的焦点，也是一种尚未得到充分研究的病原体。我们还将检测新出现的Cps病原体和非生物威胁性病原体，这些病原体是非典型肺炎的主要原因，在临床表现中经常与生物威胁性病原体混淆：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，《生物防御》RFA-AI-11-014指出，“使用平台快速区分个人感染的是生物威胁因子还是具有类似、普遍症状的普通感染的医学诊断是高度优先考虑的。”目前在美国，这些病原体的商业诊断很少或没有。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），如初步研究所示，用于从临床样品中纯化基因组DNA，扩增，电泳分离和激光检测并分型Ct。整个系统从DNA纯化到检测大约需要1小时。目的是：1)建立具有代表性的Cps生物威胁性和非生物威胁性非典型呼吸道病原体的基因组序列，以进行稳健的引物选择；2)修饰BCS以纯化临床鼻咽拭子和痰液样本中的基因组DNA；3)利用Read博士开发的引物选择管道，根据现有基因组的比较基因组学和Aim 1中测序的引物识别引物，用于区分生物威胁性和非生物威胁性非典型呼吸道病原体，并开发一种多重DNA扩增试验来区分每种病原体；4)评估NetBio检测的敏感性、特异性和阳性和阴性预测值，首先使用加标样品，然后使用临床NP拭子和痰样本，与现有的商业核酸扩增检测Ct、Cpn、Mp和Lp（不存在Cps）进行比较，并与FDA批准临床试验之前的高灵敏度内部RT-PCR检测进行比较。未来的目标是将该检测扩展到其他具有生物威胁性的呼吸道病原体。我们设想NetBio样品到答案分析将用于急诊室、医学博士办公室、诊所、军事设施、医院和现场，以促进我们的理解

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.