Developing a multiplex diagnostic test for SNPs related to dry eye disease

开发与干眼病相关的 SNP 的多重诊断测试

• 批准号: 10481411
• 负责人: DAVID A SHAFER
• 金额: $ 25.81万
• 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: 2022-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10481411
• 关键词: Adult; Affect; Alleles; Binding; Biological Assay; Biological Markers; CLIA certified; COVID-19 assay; Characteristics; Cicatrix; Clinical; Complex; DNA; Data; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Discrimination; Disease; Disease susceptibility; Doctor of Philosophy; Dry Eye Syndromes; Early Diagnosis; Etiology; Excessive tearing; Film; Fluorescence; Freeze Drying; Future; Genetic; Genomic DNA; Gold; Hospitals; Human; Individual; Inflammation; Label; Laboratories; Letters; Limes; Link; Marketing; Measures; Methods; Molecular; Molecular Diagnostic Testing; Mucin 1 protein; Nucleic Acid Amplification Tests; Osmolar Concentration; Pathway interactions; Patient Care; Patients; Performance; Phase; Physicians; Prevention; Production; Reporting; Research Contracts; Risk; Risk Factors; Routine Diagnostic Tests; Running; Sampling; Signs and Symptoms; Single Nucleotide Polymorphism; Specificity; Specimen; Symptoms; System; TNF gene; Technology; Temperature; Testing; Time; Tube; Ulcer; United States; VDR gene; Validation; base; commercialization; cost; cross reactivity; detection limit; diagnosis evaluation; diagnosis standard; diagnostic assay; diagnostic biomarker; disease diagnosis; evaporation; experience; genome wide association study; improved; interest; melting; multiplex diagnostics; novel; ocular surface; phase 2 testing; prevent; research clinical testing; risk variant; success; validation studies

Developing a multiplex diagnostic test for SNPs related to dry eye disease Confidential PI: Shafer, David A., PhD PROJECT SUMMARY: Dry eye disease (DED) is one of the most common ophthalmic conditions, affecting 1 in 5 adults worldwide and 5–15% of all adults in the United States. DED is characterized by increased tear film osmolarity and inflammation on the ocular surface, which manifests in patients as various symptoms of discomfort. A major objective of Notice of Special Interest NOT-EY-21-007 is to validate diagnostic biomarkers associated with the risk of developing DED in asymptomatic individuals. DED diagnosis is challenging due to the complex etiological mechanisms underlying the disease, a poor correlation between clinical signs and symptoms, and the lack of a gold standard for diagnosis. DED diagnosis and clinical evaluation are subjective and normally depend on patient-reported symptoms. However, diagnosis based on symptoms alone is unreliable because the symptoms of DED overlap with those of other ocular conditions. Thus, a significant need exists for reliable methods to identify individuals at risk for developing DED or to support the diagnosis of DED. Genetic factors contribute to the symptoms and signs of DED. Specifically, single-nucleotide polymorphisms (SNPs) in the IL1B, IL6R, MUC1, TNF-a, TNFAIP3, and VDR genes have been implicated as risk factors associated with DED. Therefore, developing a nucleic acid amplification test for these SNPs should help identify at-risk individuals and support DED diagnosis. GeneTAG Technology has invented several qPCR probe systems with a novel error-prevention mechanism, which provides excellent specificity. Our internal DNA-Detection Switch (iDDS) probe system comprises a fluor-labeled probe and a quencher-labeled antiprobe that is nearly complementary to the probe. In the absence of the intended target, the probe and antiprobe bind together, which quenches fluorescence and prevents false-positive results. In this study, we will generate a diagnostic test for DED risk alleles using the iDDS probe system. In Specific Aim 1, we will develop a lyophilized 8-tube dual-iDDS probe assay for SNPs linked to dry eye disease. The key milestones of Aim 1 are to (i) develop dual-iDDS probe assays for SNPs linked to DED, (ii) convert the assays to lyophilized format, and (iii) confirm the performance of each assay following lyophilization. In Specific Aim 2, we will evaluate key performance characteristics of the multiplex dry eye disease test. The Aim 2 milestones are to determine the (i) linearity, (ii) limit of detection (LoD), (iii) precision, and (iv) stability of the dual-iDDS probe assays with human genomic DNA samples, homozygous for each allele of interest. Success in Phase I will support expanded Phase II testing, which will be conducted through a contract research organization. In Phase II, expanded assay characterization (analytical reactivity, cross-reactivity, carryover, cross-contamination, assay cut-off) will be performed using clinical specimens. Project success will facilitate development of the first molecular diagnostic test for SNP alleles linked to DED susceptibility, which will support patient diagnosis and improve patient care.

针对与干眼病机密PI相关的SNP开发多重诊断测试：Shafer，David A.，PhD 项目摘要： 干眼症（DED）是最常见的眼科疾病之一，影响了全球五分之一的成年人。 在美国所有成年人中，有5-15％。 DED的特征是撕裂膜渗透压和炎症增加 在眼部表面，在患者中表现为各种不适的符号。通知的主要目标 特殊兴趣而非EY-21-007的特殊兴趣是验证与发展风险相关的诊断生物标志物 无症状的个体。由于复杂的病因机制，DED诊断很具有挑战性 疾病的根本，临床体征和症状之间的相关性差，缺乏黄金标准 DED诊断和临床评估是主观的，通常取决于患者报告 症状。但是，仅基于症状的诊断是不可靠的，因为DED重叠的症状 与其他眼部条件的情况。这是对可靠方法识别个人的重要需求 有开发DED或支持DED诊断的风险。遗传因素有助于符号和 ded的迹象。具体而言，IL1B，IL6R，MUC1，TNF-A，TNFAIP3的单核苷酸多态性（SNP） VDR基因已被暗示为与DED相关的风险因素。因此，开发核酸 这些SNP的放大测试应有助于识别高危个人并支持DED诊断。 Genetag 技术已经发明了一些具有新颖的误差机制的QPCR探针系统，该机制提供了 出色的特异性。我们的内部DNA检测开关（IDDS）探针系统包括荧光标记的探针 以及几乎完整的探针的淬灭剂标记的抗植物。在没有意图的情况下 靶，探针和抗果杆结合在一起，从而释放荧光并防止假阳性结果。 在这项研究中，我们将使用IDDS探测系统生成用于DED风险等位基因的诊断测试。在特定目标中 1，我们将开发一个冻干的8块双IDDS探测测定，用于与干眼症相关的SNP。钥匙 AIM 1的里程碑是（i）开发链接到DED的SNP的双IDDS探测测定，（ii）转换测定法 冻干格式，（iii）确认冻干后每种测定的性能。在特定的目标2中， 我们将评估多重眼科疾病测试的关键性能特征。目标2里程碑是 确定（i）线性，（ii）检测极限（lod），（iii）精度和（iv）双IDD的稳定性证明 人类基因组DNA样品的测定，对于每个感兴趣的等位基因纯合子。第一阶段的成功将 支持扩展的II期测试，该测试将通过合同研究组织进行。在阶段 II，扩展的测定表征（分析反应性，交叉反应性，结转性，交叉污染，测定 截止）将使用临床标本进行。项目成功将有助于开发第一个 与DED敏感性相关的SNP等位基因的分子诊断测试，这将支持患者诊断和 改善患者护理。