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）：尽管抗逆转录病毒治疗长期抑制了循环病毒，但HIV-1储库仍以潜伏形式存在。实现治愈HIV-1感染的主要挑战是这些潜伏病毒储库的持续存在。测定 需要允许鉴定、表征和分离潜伏感染的单细胞用于下游基因组测序的方法来有效和完全表征HIV-1储库。然而，分析潜伏感染细胞的现有测定需要繁琐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在昂贵的设备和试剂上进行大量投资，并且尚未适用于潜在储库的下游表征。我们建议使用微流控方法和PCR开发和验证一种创新的新型检测方法，用于对潜伏感染的人类细胞中的病毒基因组进行识别、计数、分离和下游表征。我们的方法的应用将是特别有用的组合抗逆转录病毒治疗和/或研究的新模式的水库根除患者的样本的分析。具体目标包括：1）开发和测试所提出的方法的功效，以使用微流体方法和PCR来鉴定和计数潜伏感染的人PBMC、组织来源的巨噬细胞和其他原代人细胞，以及2）验证我们的测定以分离具有整合的HIV-1 DNA的单细胞液滴用于下游测序和次级靶基因定量。 这项为期两年的发展赠款将利用创新方法和现有微流控技术的调整，开发一种检测方法，以在抗逆转录病毒治疗患者的单细胞水平上表征艾滋病毒储库。我们的建议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。我们先前在检测和定量非常低水平的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将对以下方面至关重要： 开发新的检测方法，在单细胞水平上识别和表征HIV感染。所提出的方法有可能适用于各种各样的多学科研究，如单细胞表征的病毒和细胞内 病原体或干细胞和/或恶性组织的分析。