Next Generation Infectious Disease Diagnostics: Microfluidic-Free Gigapixel PCR with Self-Assembled Partitioning

下一代传染病诊断：具有自组装分区的无微流控千兆像素 PCR

基本信息

批准号：
10682295
负责人：
Adam R. Abate
金额：
$ 62.37万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-15 至 2028-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10682295
关键词：
Address Bacterial Infections Biological Assay Capillary Electrophoresis Central Nervous System Bacterial Infections Central Nervous System Infections Clinic Clinical Communicable Diseases Complex Concentration measurement Custom Cytomegalovirus Detection Diagnosis Diagnostic Dideoxy Chain Termination DNA Sequencing Emulsions Equipment FDA approved Family Flaviviridae Flavivirus Infections Generations HIV Health Hepatitis C Hospitals Human Resources Individual Infection Investments Laboratories Length Licensing Liquid substance Manuals Measurement Measures Meningitis Methods Microfluidics Minor Molecular Nature Nosocomial Infections Nucleic Acids Nucleotides Organism Patient-Focused Outcomes Patients Persons Preparation Procedures Protocols documentation Reaction Reporting Reproducibility Resolution Respiratory Tract Infections Robotics Running Sampling Sepsis Shipping Source Specialist Symptoms Techniques Technology Testing Time Tube Variant Virus Diseases Vision West Nile virus Work Zika Virus accurate diagnosis accurate diagnostics arm clinical diagnostics cost digital disease diagnostic gastrointestinal infection human genome sequencing innovation instrument multiplex assay multiplex detection next generation novel nucleic acid quantitation pathogen ratiometric respiratory respiratory virus self assembly skills technology validation treatment response

项目摘要

ABSTRACT Infections by different pathogens can manifest with similar symptoms, but appropriate treatment requires specific and accurate diagnosis. Clinicians often turn to multiplexed assays testing for many organisms (e.g. BioFire). While these approaches can test for 50-70 organisms, they do not provide concentration titers, which is necessary to identify the causative pathogen among the several false positives or clinically meaningless commensals. As a result, the clinician must perform additional tests to identify which of the positives is causative. Although these tests use quantitative PCR, in clinical labs the results are reported as presence/absence due to the finicky nature of PCR in this setting, which is sensitive to minor variations in reaction efficiency, operator variability. As a result, today, only a few widespread PCR tests are FDA approved to report quantitative result. In contrast to qPCR, digital PCR (dPCR) measures target titers by counting individual molecules. As a result, dPCR provides an absolute concentration measurement that doesn’t require a standard curve. In addition, the reaction is cycled to endpoint, then quantified; it does not require careful estimation of the amplification rate, which is a major source of variability in qPCR. Thus, dPCR is less sensitive to variations in reaction efficiency and provides superior consistency. However, current dPCR methods are limited in multiplexing, allowing just 5- 6 targets per assay, while qPCR can test up to 100. Moreover, dPCR requires complex microfluidic equipment that burdens testing lab personnel and increases cost. Until these issues can be addressed, qPCR will continue to dominate the clinical lab, and quantitative and absolute pathogen load reporting will remain beyond reach. Here, we propose a novel nucleic acid technology combining the quantitativeness and robustness of dPCR with the simplicity and multiplexing of qPCR. Our vision is to enable broad spectrum detection wherein each pathogen is associated with a high confidence, quantitative titer. Our approach – gigapixel PCR (gPCR) – is enabled by our recent discoveries of self-assembled partitioning, for microfluidic-free generation of monodispersed emulsions, and linearized target quantitation with capillary electrophoresis (CE). CE allows sensitive quantitation over 7 decades and provides amplicon length information with single nucleotide resolution. In gPCR, we use this to perform multiplexed detection of over 100 amplicons per reaction. In contrast to qPCR, which requires that the sample be split to test for different targets, thereby diluting it and reducing sensitivity, with gPCR the targets are tested without splitting, maintaining them at maximal concentration, and substantially increasing sensitivity. Moreover, based on robust dPCR, gPCR provides reproducible, quantitative results across testing conditions. It thus addresses the major limitations of current dPCR technologies and provides the first viable alternative to qPCR in the clinic. We will develop and validate the technology against accepted standards (SeraCare), and work with our longstanding collaborators (Drs. Melanie Ott and Charles Chiu) to apply it to respiratory and CNS infections from samples previously collected at UCSF hospitals.

抽象的不同病原体感染可表现出相似的症状，但适当的治疗需要具体的治疗和准确的诊断。临床医生经常求助于多种生物体的多重检测（例如 BioFire）。虽然这些方法可以测试 50-70 种生物体，但它们不提供浓度滴度，这是需要在几种假阳性中找出致病病原体或无临床意义因此，临床医生必须进行额外的测试来确定哪些阳性结果是致病的。尽管这些测试使用定量 PCR，但在临床实验室中，由于以下原因，结果会报告为存在/不存在： PCR 在这种情况下的挑剔性质，对反应效率、操作员的微小变化很敏感因此，如今，只有少数广泛使用的 PCR 检测获得 FDA 批准来报告定量结果。与 qPCR 不同，数字 PCR (dPCR) 通过计数单个分子来测量目标滴度。 dPCR 提供不需要标准曲线的绝对浓度测量。反应循环至终点，然后定量；不需要仔细估计扩增率，这是 qPCR 变异的主要来源，因此，dPCR 对反应效率的变化不太敏感。并提供卓越的一致性。然而，当前的 dPCR 方法在多重检测方面受到限制，仅允许 5- 每次检测可检测 6 个目标，而 qPCR 最多可检测 100 个目标。此外，dPCR 需要复杂的微流体设备在这些问题得到解决之前，qPCR 将继续下去。主导临床实验室，定量和绝对病原体负荷报告仍然遥不可及。在这里，我们提出了一种新颖的核酸技术，将 dPCR 的定量性和稳健性与 qPCR 的简单性和多重性我们的愿景是实现每种病原体的广谱检测。与高置信度、定量滴度相关，我们的方法——十亿像素 PCR (gPCR)——是通过以下方式实现的。我们最近发现的自组装分配，用于无微流体生成单分散乳液和毛细管电泳 (CE) 线性化目标定量可实现灵敏定量。超过 7 个十年，并提供单核苷酸分辨率的扩增子长度信息，在 gPCR 中，我们使用它。每个反应可对超过 100 个扩增子进行多重检测，而 qPCR 则需要这样做。将样品分开以测试不同的目标，从而稀释样品并降低灵敏度，使用 gPCR 检测目标在不分裂的情况下进行测试，将它们保持在最大浓度，并显着提高灵敏度。此外，基于强大的 dPCR，gPCR 可在各种测试条件下提供可重复的定量结果。因此解决了当前 dPCR 技术的主要局限性，并提供了第一个可行的替代方案我们将根据公认的标准 (SeraCare) 开发和验证该技术，以及与我们的长期合作者（Melanie Ott 博士和 Charles Chiu 博士）合作，将其应用于呼吸系统和中枢神经系统来自先前在加州大学旧金山分校医院收集的样本的感染。