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.

双idds探针多重检测方法在地方性真菌病原体检测中的应用[j]