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

• 批准号: 10591947
• 负责人: DAVID A SHAFER
• 金额: $ 20.7万
• 依托单位: GENETAG TECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-06 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10591947
• 关键词: Acute Respiratory Distress Syndrome; Adult; Amphotericin B; Anatomy; Aspergillus; Biological Assay; Blastomyces; Blastomyces dermatitidis; Blastomycosis; CLIA certified; Candida; Certification; Cessation of life; Characteristics; Child; Chronic; Clinical; Clinical Pathology; Coccidioides; Coccidioides immitis; Coccidioides posadasii; Coccidioidomycosis; Communicable Diseases; Cryptococcus; DNA; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Differential Diagnosis; Disease; Doctor of Philosophy; Drug resistance; Ensure; FDA approved; Fluconazole; Fluorescence; Freeze Drying; Future; Health Benefit; Healthcare; Histoplasma; Histoplasma capsulatum; Histoplasmosis; Hospitals; Immunocompetent; Immunocompromised Host; Individual; Infection; Intervention; Itraconazole; Label; Laboratories; Life; Liquid substance; Lung diseases; Lymphoma; Malignant Neoplasms; Malignant neoplasm of lung; Manufacturer; Marketing; Medical; Meningeal; Methods; Molecular; Molecular Diagnostic Testing; Monitor; Morbidity - disease rate; Mycoses; Pathway interactions; Patients; Performance; Pericarditis; Phase; Physicians; Pneumonia; Public Health; Pulmonary Coin Lesion; Rationalization; Reflex action; Reporting; Research Contracts; Resistance; Routine Diagnostic Tests; Sampling; Sarcoidosis; Serology; Site; Specialist; Specificity; Specimen; Symptoms; System; Technology; Test Result; Testing; Thoracic Radiography; Time; Tube; Validation; accurate diagnosis; color detection; commercialization; cost; cross reactivity; desert fever; design; detection limit; diagnostic assay; effective therapy; experience; flu; instrument; locked nucleic acid; mortality; multiplex diagnostics; pathogen; patient subsets; prevent; resistance mutation; respiratory; success

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.

开发双IDDS探针多路复用测试，用于检测内部分子真菌剂机密PI：Shafer，David A.，PhD 项目摘要 在这项研究中，我们提出了开发一种简单的，冻干的分子检测，以检测 山谷热（球虫菌病），组织胞浆症或胚菌病，基于我们的专有内部DNA- 检测开关（IDD）探针技术。这些真菌感染的及时诊断是选择的关键 有效治疗并预防严重的发病率或死亡。认为超过500,000个这样的案件发生在 每年美国，尽管仅存在一次FDA批准的分子测试，以供诊断山谷发烧，而没有FDA- 存在批准的分子检测，用于诊断性组织胞浆症或爆术。 球虫菌病，组织胞浆症和爆术的症状经常投票和胸部X射线 不要区分这些和其他肺部疾病。那就是这些疾病之间的鉴别诊断 还有其他症状（急性呼吸窘迫综合征，肺癌，淋巴瘤， 心包炎，结节病或固体肺结核）是主动医疗干预措施的关键。而且， 使用基于培养或血清学方法的诊断可能会有数周或数月的周转时间，或者显示 假阳性结果和唯一相关的FDA批准测试（genestat.mdx球虫下测定）仅是 被批准用于制造商的仪器，该仪器劝阻在参考实验室使用。 Genetag技术（www.genetagtech.com）专门开发敏感且可靠的QPCR探针 系统。我们的防误IDDS探针系统采用荧光标记的目标特定探针和A 略微不匹配的淬灭剂抗果。在没有预期目标的情况下，抗果实杂交 进行探测，淬灭其荧光并防止脱靶检测。我们还开发了双idds 探针测定提高特异性和自动确认测试结果。这增加了 特异性是准确诊断的关键，尤其是在真菌感染水平较低的情况下。冗余检测 多个目标站点大大减少了对反射/验证性测试的需求。我们的具体目的是（1） 三台双IDDS探测分析，用于immisis，C。posadasii，H。capsulatum和B. dermatitidis，以及（2） 评估冻干格式内粒真菌剂的多重测试的关键性能特征。 具体而言，在AIM 1中，我们将（i）开发锁定的核酸（LNA）增强双IDDS探针以进行诊断 每种病原体中的序列特异性靶标，（ii）将双IDD问题转换为冻干格式，以及（iii） 用冻干测定进行交叉反应性测试，以评估其他病原体的假阳性检测 通常在支气管肺泡液中发现。在AIM 2中，我们将确定（i）线性，（ii）检测极限（LOD）， （iii）精度，（iv）干扰物质（如果有）和（v）双IDDS探测测定的稳定性的影响。 在这项工作中的成功将通过BSL III-在II阶段的临床标本中证明扩大的测试合理 认证合同研究组织。