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

• 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
• 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

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）：尽管长期抑制了抗逆转录病毒疗法的循环病毒，但HIV-1储层仍以潜在形式存在。实现HIV-1感染的治疗方法的主要挑战是这些潜在病毒储层的持久性。测定 需要鉴定，表征和隔离潜在感染的单细胞FO下游基因组测序，以有效并充分表征HIV-1储层。但是，分析潜在感染细胞的现有测定需要繁重且昂贵的连续细胞稀释液。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，并且尚未适应潜在储层的下游表征。我们建议使用具有PCR的微流体方法来开发和验证一种创新和新颖的测定法，以鉴定，枚举以及分离和下游表征来自潜在受感染的人类细胞的病毒基因组。我们方法的应用将在分析抗逆转录病毒疗法的患者和/或新型储层消除方式研究中的样本中特别有用。具体目的包括：1）使用微流体方法和PCR识别和枚举潜在的人类PBMC，组织衍生的巨噬细胞以及其他原代人细胞的拟议方法的功效，以及2）2）验证我们的实现我们的单细胞液滴，以隔离HIV-1 DNA隔离HIV-1 DNA，以实现下流的量级量化，并获得了量子的量级。 这项为期两年的开发赠款将利用现有微流体技术的创新方法和适应性，以开发一种在抗逆转录病毒治疗患者的单细胞水平上表征HIV-储能的测定法。我们的建议涉及具有不同但免费研究背景和经验的主要研究人员，包括翻译病毒学和生物工程/生物物理学。我们先前在检测和定量非常低的HIV-1遗传材料以及开发用于病毒和免疫特征的微流体设备方面的经验对 在单细胞水平上识别和表征HIV感染的新型测定法的开发。所提出的方法有可能适应多种多学科研究，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。