Microfluidic PCR Method to Identify and Characterize HIV-Infected Single Cells

微流控 PCR 方法鉴定和表征 HIV 感染的单细胞

基本信息

批准号：
8790281
负责人：
Utkan Demirci
金额：
$ 22.62万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8790281
关键词：
3-Dimensional Biological Assay Biomedical Engineering Biophysics Blood specimen CD4 Positive T Lymphocytes Cancer Biology Cell Count Cell Line Cells Chromosomes Clinical Cytolysis DNA Detection Development Disseminated Malignant Neoplasm Emulsions Encapsulated Equipment Frequencies Future Gene Targeting Genes Genetic Materials Genome Genomic DNA Genomics Grant HIV HIV Infections HIV-1 Human Human Genome Immune Individual Infection Interdisciplinary Study Investigation Investments Laboratories Length Liver Lymphocyte Malignant - descriptor Malignant neoplasm of ovary Methods Microfluidic Microchips Microfluidics Modality Mononuclear Neoplasm Circulating Cells Nodule Patients Performance Peripheral Blood Mononuclear Cell Principal Investigator Publishing RNA Reaction Reagent Recovery Research Rest Running Sampling Sensitivity and Specificity Sorting - Cell Movement Stem cells Surveys System Techniques Technology Testing Tissue Embedding Tissue Sample Tissues Viral Viral Genome Virus antiretroviral therapy design digital efficacy testing experience flexibility genome sequencing high throughput screening innovation insight macrophage nanolitre scale nanoscale novel pathogen peripheral blood public health relevance research study screening single cell analysis virology

项目摘要

DESCRIPTION (provided by applicant): HIV-1 reservoirs continue to exist in latent form despite long-term suppression of circulating virus with antiretroviral therapy. The main challenge in achieving a cure for HIV-1 infection is the persistence of these latent viral reservoirs. Assays that allow for identification, characterization, and isolation of latently infected single cells fo downstream genomic sequencing are needed to efficiently and fully characterize HIV-1 reservoirs. However, existing assays that analyze latently-infected cells require burdensome and costly serial cell dilutions. Other proposed methods to identify and analyze HIV-infected cells require significant investment in costly equipment and reagents and have not been adapted for downstream characterization of latent reservoirs. We propose to develop and validate an innovative and novel assay using microfluidic methods with PCR for identification, enumeration, and isolation and downstream characterization of viral genomes from latently-infected human cells. The application of our approach will be particular useful in the analysis of samples from patients on combination antiretroviral therapy and/or in studies of novel modalities of reservoir eradication. Specific aims include: 1) develop and test the efficacy of the proposed method to identify and enumerate latently-infected human PBMCs, tissue derived macrophages, and other primary human cells using microfluidic methods and PCR, and, 2) validate our assay to isolate single-cell droplets with integrated HIV-1 DNA for downstream sequencing and secondary target gene quantification. This two-year development grant will utilize innovative approaches and adaptations of existing microfluidic technologies to develop an assay to characterize HIV- reservoirs on the single-cell level in patients on antiretroviral therapy. Our proposal involves principal investigators with different but complimentary research backgrounds and experiences, including translational virology and bioengineering/biophysics. Our prior experiences in the detection and quantification of very low-levels of HIV-1 genetic material and in the development of microfluidic devices for viral and immune characterization will be crucial to the development of novel assays to identify and characterize HIV-infection at the single-cell level. The proposed method has the potential to be adapted for a wide variety of multidisciplinary research studies, such as single-cell characterization of viral and intracellular pathogens or analysis of stem cells and/or malignant tissues.

描述（由申请人提供）：尽管通过抗逆转录病毒疗法长期抑制了循环病毒，但 HIV-1 病毒库仍以潜伏形式存在。治愈 HIV-1 感染的主要挑战是这些潜伏病毒库的持续存在。化验需要能够识别、表征和分离潜伏感染的单细胞以进行下游基因组测序，以有效、全面地表征 HIV-1 病毒库。然而，现有的分析潜伏感染细胞的分析方法需要繁重且昂贵的连续细胞稀释。其他提出的识别和分析 HIV 感染细胞的方法需要对昂贵的设备和试剂进行大量投资，并且尚未适用于潜在储存库的下游表征。我们建议开发和验证一种创新和新颖的检测方法，使用微流体方法和 PCR 来识别、计数、分离和下游表征来自潜伏感染的人类细胞的病毒基因组。我们的方法的应用对于分析接受联合抗逆转录病毒治疗的患者样本和/或研究新的病毒库根除方式特别有用。具体目标包括：1) 开发和测试所提出的方法的有效性，以使用微流体方法和 PCR 来识别和计数潜伏感染的人类 PBMC、组织来源的巨噬细胞和其他原代人类细胞，以及 2) 验证我们的检测方法以分离单个-带有集成 HIV-1 DNA 的细胞液滴，用于下游测序和二级靶基因定量。这笔为期两年的开发补助金将利用创新方法和现有微流体技术的改进来开发一种分析方法，以在接受抗逆转录病毒治疗的患者的单细胞水平上表征HIV病毒库。我们的提案涉及具有不同但互补的研究背景和经验的主要研究人员，包括转化病毒学和生物工程/生物物理学。我们之前在检测和定量极低水平的 HIV-1 遗传物质以及开发用于病毒和免疫表征的微流体装置方面的经验对于开发新的检测方法以在单细胞水平上识别和表征艾滋病毒感染。所提出的方法有可能适用于各种多学科研究，例如病毒和细胞内的单细胞表征病原体或干细胞和/或恶性组织的分析。