Device for Preservation of Cell Free RNA in Saliva

唾液中游离 RNA 的保存装置

• 批准号: 10417132
• 负责人: Shivani Nautiyal
• 金额: $ 22.78万
• 依托单位: PRIME GENOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-03 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10417132
• 关键词: Adoption; Automobile Driving; Biocide; Biological Assay; Biological Markers; Blood specimen; Breast Cancer Detection; COVID-19; COVID-19 detection; COVID-19 pandemic; Cells; Centrifugation; Characteristics; Childhood; Clinic; Clinic Visits; Clinical; Clinical Research; Collection; Complex; Cytolysis; DNA; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic tests; Disadvantaged; Disease; Early Diagnosis; Facility Accesses; Feedback; Fractionation; Freezing; Future; Genomics; Grant; Growth; Health; Healthcare; Home; Hour; Human; Image; Individual; Laboratories; Life; Link; Malignant Neoplasms; Manuals; Measurement; Measures; Medical; Medicine; Membrane; Methods; Microbe; Mixed Cellular Population; Molecular; Monitor; Noise; Nucleic Acids; Office Visits; Participant; Patients; Performance; Phase; Population; Process; Property; Quarantine; RNA; RNA Decay; RNA Degradation; RNA Stability; RNA marker; Reagent; Recovery; Research; Saliva; Salivary; Sampling; Savings; Ships; Site; Small Business Innovation Research Grant; Standardization; Surveillance Program; Systemic disease; Temperature; Testing; TimeLine; Training; Traumatic Brain Injury; Validation; Venous blood sampling; Viral; Virus; Woman; Work; autism spectrum disorder; base; biomarker panel; breast imaging; cancer diagnosis; clinical diagnostics; cost; design; exosome; experimental study; innovation; iterative design; malignant breast neoplasm; microbial; microbiota; microorganism growth; mortality; nasal swab; next generation sequencing; nuclease; oral microbial community; oral microbiome; pandemic disease; participant enrollment; pathogen; point of care; post-COVID-19; preservation; prevent; programs; prospective; protein metabolite; prototype; saliva sample; salivary assay; salivary cell; sample collection; screening; trait; usability; virtual

ABSTRACT Early detection is critical to cancer mortality reductions, and yet the breast imaging clinic visits required for these life-saving early detection programs are inaccessible to disadvantaged, remote, or quarantined populations with limited medical facility access. In fact, breast cancer surveillance programs this year have stalled by as much as 90% versus their pre-pandemic rates, resulting in risky delays in new cancer diagnosis [1]. A saliva-based test for early detection could transform today’s screening paradigms. A promising biofluid for disease detection, saliva can be self-sampled non-invasively, without trained technicians. Saliva is ideal for at-home and other non-clinical site collection; it contains a variety of analytes including exosomes, RNA, DNA, proteins, metabolites, viruses and oral microflora, and is a powerful window on health and disease. Recently, saliva samples have shown better clinical utility vs. nasal swabs for detecting COVID-19 [2]. Mounting evidence exists for oral microbiome links to systemic disease [3], and salivary biomarkers diagnose complex traits such as pediatric medical conditions, traumatic brain injury, autism, and cancer [4-7]. A commercial saliva-based test for autism is based on 32 human and microbial RNA markers [8]. In many complex trait studies, saliva is frozen as cell-free supernatant. The most prevalent saliva collection and preservation devices, although easy-to-use, preclude effective cell-free RNA and exosome analysis, since saliva samples are treated with lysis reagents resulting in mixed populations of cellular and non-cellular RNAs, increasing noise and damaging performance characteristics. Saliva collection devices that instead use membranes to separate cells suffer from inefficient recovery of critical analytes, either because they lack appropriate preservatives or because they adsorb and therefore lose, a substantial analyte fraction. To enable widespread use for disease detection, new standardized methods are needed to preserve cell-free saliva and retain the integrity of its key constituents for stable transport and downstream analysis. This project extends our past SBIR grant work and our own internal research, to develop an easy-to-use saliva collection and preservation device to stabilize cfRNA and other analytes, enabling us and others to efficiently collect high-quality clinical saliva samples. Using RT-qPCR assays and RNA isotype profiles, we will characterize salivary cfRNA integrity and stability over timelines sufficient for remote collection and transport. Armed with well-characterized RNA decay profiles, we propose an iterative design-of-experiment approach to prototype a versatile device to collect and preserve saliva at room temperature (RT), stabilizing RNA while also preventing cell lysis and bacterial growth. Our device performance validation will use prospectively collected and preserved saliva from >50 women to demonstrate stability for up 72 hours at ambient conditions. This device will standardize methods for collection, stability, and transport of saliva, key to driving virtualized sample collection and healthcare in cancer, viral, and other diagnostics, increasingly critical to a post-pandemic world.

抽象的 早期检测对于降低癌症死亡率至关重要，但是乳房成像诊所就诊 这些挽救生命的早期检测计划无法访问灾难，遥远或隔离 医疗设施有限的人群。实际上，今年乳腺癌监测计划已有 与其大流行前率相比，停滞不前的速度高达90％，导致新的癌症诊断延迟 [1]。基于唾液的早期检测测试可以改变当今的筛查范例。有希望的生物流体 为了疾病检测，无训练的技术人员，唾液可以非侵入性地自杀。唾液是理想的选择 在家和其他非临床场地集合；它包含各种分析物，包括外泌体，RNA，DNA， 蛋白质，代谢产物，病毒和口腔菌群，是健康和疾病的强大窗口。最近， 唾液样品显示出更好的临床效用与用于检测COVID-19的鼻拭子[2]。安装 存在与全身性疾病的口服微生物组联系的证据[3]，唾液生物标志物诊断复合物 小儿医疗状况，脑损伤，自闭症和癌症等特征[4-7]。广告 基于唾液的自闭症测试基于32个人和微生物RNA标记[8]。在许多复杂的特征中 研究，唾液被冷冻为无细胞上清液。最普遍的唾液收藏和保存 设备虽然易于使用，但由于唾液样品而排除了有效的无细胞RNA和外泌体分析 用裂解试剂处理，导致细胞和非细胞RNA混合种群，增加 噪声和破坏性性能特征。唾液收集设备，而不是使用机制 单独的细胞遭受关键分析物效率低下的恢复，要么是因为它们缺乏合适的 防腐剂或因为它们吸附并因此损失，这是一个大量的分析物。启用宽度 用于疾病检测，需要新的标准化方法来保留无细胞的唾液并保留 其关键的完整性构成稳定的运输和下游分析。这个项目扩展了我们的过去 Sbir Grant的工作和我们自己的内部研究，以开发易于使用的唾液收集和保存 稳定CFRNA和其他分析物的设备，使我们和其他人能够有效收集高质量的临床 唾液样品。使用RT-QPCR分析和RNA同型曲线，我们将表征唾液CFRNA完整性 和时间表上的稳定性足以容纳远程收集和运输。配备良好的RNA 衰减轮廓，我们提出了一种迭代实验设计方法，用于原型一种多功能设备来收集 并在室温下保存唾液（RT），稳定RNA，同时还可以防止细胞裂解和细菌 生长。我们的设备性能验证将使用前瞻性收集和保存的唾液> 50 在环境条件下，妇女表现出72小时的稳定性。该设备将标准化方法 唾液的收集，稳定和运输，是推动虚拟化样本收集和医疗保健的关键 癌症，病毒和其他诊断，对大流行世界越来越重要。