Development of a dual-iDDS probe multiplex test for detecting endemic fungal agents

开发用于检测地方性真菌因子的双iDDS探针多重测试机密PI：Shafer，大卫A.，博士 项目摘要 在这项研究中，我们建议开发一种简单的，冻干的分子检测检测病原体的 山谷热（球孢子菌病），组织胞浆菌病，或芽生菌病，根据我们专有的内部DNA- 检测开关（iDDS）探头技术。及时诊断这些真菌感染是选择的关键 有效治疗和预防严重发病或死亡。据认为，在非洲， 美国每年，虽然只有一个FDA批准的分子测试存在诊断山谷热，没有FDA- 存在用于诊断组织胞浆菌病或芽生菌病的批准的分子测试。 球孢子菌病、组织胞浆菌病和芽生菌病的症状常常模糊不清， 不能区分这些和其他肺部疾病。因此，这些疾病之间的鉴别诊断 和其他表现出类似症状（急性呼吸窘迫综合征，肺癌，淋巴瘤， 心包炎、结节病或孤立性肺结节）是积极医疗干预的关键。此外，委员会认为， 使用基于培养或血清学的方法进行诊断可能需要数周或数月的周转时间， 假阳性结果，而唯一相关的FDA批准的检测（GeneSTAT.MDx球孢子菌检测）仅 批准在制造商的仪器上使用，这不鼓励在参考实验室使用。 GeneTAG Technology（www.genetagtech.com）专注于开发灵敏可靠的qPCR探针 系统.我们的抗错误iDDS探针系统采用荧光标记的靶特异性探针和荧光标记的靶特异性探针。 轻微错配的淬灭剂标记的反探针。在没有预期靶点的情况下， 使其荧光猝灭并防止脱靶检测。我们还开发了双iDDS 探针检测可提高检测结果的特异性和自动确认。这一层 特异性是准确诊断的关键，特别是对于低水平的真菌感染。冗余检测. 多个目标部位大大减少了对反射/确认测试的需要。我们的目标是：（1）发展 3管双iDDS探针法检测C. immitis，C. posadasii，黑腹蛛H. capsulatum和B.皮肤炎，和（2）至 评价冻干形式地方性真菌病原体多重检测的关键性能特征。 具体而言，在目标1中，我们将（i）开发用于诊断的锁核酸（LNA）增强的双iDDS探针， 每种病原体中的序列特异性靶标，（ii）将双iDDS探针转化为冻干形式，和（iii） 使用冻干检测试剂进行交叉反应性试验，以评估其他病原体的假阳性检测 常见于支气管肺泡液中在目标2中，我们将确定（i）线性，（ii）检测限（LoD）， (iii)精密度，（iv）干扰物质（如有）的影响，以及（v）双iDDS探针测定的稳定性。 这一奋进的成功将证明通过BSL III在II期临床标本中进行扩展试验是合理的- 认证的合同研究组织。

Development of a dual-iDDS probe multiplex test for detecting endemic fungal agents Confidential PI: Shafer, David A., PhD PROJECT SUMMARY In this study, we propose developing a simple, lyophilized molecular test for detecting the causative agents of Valley fever (coccidioidomycosis), histoplasmosis, or blastomycosis, based on our proprietary internal DNA- Detection Switch (iDDS) probe technology. Timely diagnosis of these fungal infections is key for selecting effective treatments and preventing severe morbidity or death. Over 500,000 such cases are thought to occur in the US annually, although only one FDA-approved molecular test exists for diagnosing Valley fever, and no FDA- approved molecular tests exist for diagnosing histoplasmosis or blastomycosis. The symptoms of coccidioidomycosis, histoplasmosis, and blastomycosis are frequently vague and chest X-rays do not distinguish between these and other lung diseases. Thus, differential diagnosis between these diseases and other that present with similar symptoms (acute respiratory distress syndrome, lung cancer, lymphoma, pericarditis, sarcoidosis, or solitary pulmonary nodules) is key for proactive healthcare interventions. Moreover, diagnosis using culture- or serology-based methods may have a turnaround time of weeks or months, or show false-positive results, and the only related FDA-approved test (the GeneSTAT.MDx Coccidioides Assay) is only approved for use on the manufacturer’s instrument, which dissuades from use at reference labs. GeneTAG Technology (www.genetagtech.com) specializes in developing sensitive and reliable qPCR probe systems. Our error-resistant iDDS probe system employs a fluorescently labeled target specific probe and a slightly mismatched quencher-labeled antiprobe. In the absence of the intended target, the antiprobe hybridizes to the probe, quenching its fluorescence and preventing off-target detection. We have also developed dual-iDDS probe assays for increased specificity and automatic confirmation of the test results. This added layer of specificity is key for accurate diagnosis, especially with low levels of fungal infection. Redundant detection at multiple target sites greatly reduces the need for reflex/confirmatory testing. Our Specific Aims are (1) to develop a 3-tube dual-iDDS probe assay for C. immitis, C. posadasii, H. capsulatum, and B. dermatitidis, and (2) to evaluate key performance characteristics of the multiplex test for endemic fungal agents in lyophilized format. Specifically, in Aim 1, we will (i) develop locked nucleic acid (LNA)-enhanced dual-iDDS probes for diagnostic sequence-specific targets in each pathogen, (ii) convert the dual-iDDS probes to lyophilized format, and (iii) conduct cross-reactivity testing with the lyophilized assays to assess false-positive detection of other pathogens commonly found in bronchoalveolar fluid. In Aim 2, we will determine the (i) linearity, (ii) limit of detection (LoD), (iii) precision, (iv) effects of interfering substances (if any), and (v) stability of the dual-iDDS probe assays. Success in this endeavor will justify expanded testing with clinical specimens in Phase II through a BSL III- certified contract research organization.