Single Cell Diagnosis of Leukemia using Multiplex Nested PCR System

使用多重巢式 PCR 系统单细胞诊断白血病

基本信息

批准号：
7798761
负责人：
Gao Chen
金额：
$ 30.01万
依托单位：
MAXWELL SENSORS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7798761
关键词：
Actins Acute Lymphocytic Leukemia Acute Promyelocytic Leukemia Base Ratios Biological Assay Biological Models Biomedical Engineering Blood Blood specimen Bone Marrow Bone marrow biopsy California Cancer Diagnostics Cell Count Cell Line Cells Chromosomal translocation Chromosome abnormality Chronic Chronic Myeloid Leukemia Cities Clinical Collaborations Complementary DNA Complete Blood Count Computer software Cytogenetics DNA Sequence Rearrangement Data Detection Development Devices Diagnosis Diagnostic Disease Elements Emulsions Encapsulated Enzymes Evaluation Felis catus Figs - dietary Fluorescence Fluorescent Dyes Fluorescent in Situ Hybridization Fostering Generations Genes Genetic Materials Genome Germ Cells Goals HL-60 Cells HL60 Halogens Heating Hematologic Neoplasms Human Human Cell Line Image Individual K-562 K562 Cells Lead Liquid substance Malignant Neoplasms Measures Messenger RNA Methods Microfluidics Microscope Molecular Profiling Monitor Morphology Myelogenous Myeloid Leukemia Nanotechnology Nested PCR Oils Optics Patient Monitoring Patients Performance Phase Phase I Clinical Trials Philadelphia Philadelphia Chromosome Philadelphia Chromosome Positive Chronic Myelogenous Leukemia Process Protocols documentation Pump RNA-Directed DNA Polymerase Radial Reaction Reagent Residual Neoplasm Reverse Transcriptase Polymerase Chain Reaction Sampling Screening procedure Selection for Treatments Sensitivity and Specificity Signal Transduction Sorting - Cell Movement Source Specificity Speed Syringes System Technology Temperature Testing Time Transcript Tungsten Universities Width Work aqueous base bcr-abl Fusion Proteins cancer diagnosis clinical Diagnosis comparative genomic hybridization design detector disease diagnosis erythroid differentiation fusion gene granulocyte improved leukemia nanoDroplet neoplastic cell nucleic acid detection operation prognostic programs prototype public health relevance research study response sensor single cell analysis success tumor water flow

项目摘要

DESCRIPTION (provided by applicant): Single Cell Diagnosis of Leukemia using Multiplex Nested PCR System. To better direct targeted diagnostics and therapies to cancer, such as leukemia, there is a need for a simple blood-based assay that can provide expression information on a consistent basis with minimal patient discomfort. Current methods for chronic myelogenous (or myeloid) leukemia (CML) detection are based on the complete blood count, which shows increased granulocytes of all types. A bone marrow biopsy to screen for the Philadelphia chromosome is often performed as part of the evaluation for CML, but bone marrow morphology alone is insufficient to diagnose CML. In order to choose the best treatment and monitor the patient's response, it is important to know if the patient's type of leukemia is CML. To confirm the diagnosis, clinicians need to rely on a multiplex qRT-PCR assay using genes known to differentially express in express in different types of leukemia However, all current assay methods are laborious and time consuming, and no high throughput method exists for screening a large number of cells in blood samples. Therefore, Maxwell Sensors Inc. proposes to develop a Single Cell Diagnosis of Leukemia using the Nanodroplet based Multiplex Nested PCR (NMNP) System, which has the capacity to process thousand of individual cells; detect circulating tumor cells based on multiplexed PCR results; and analyze the presence or absence of chimeric BCR/ABL mRNAs for chronic myeloid leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL). The NMNP system automates all the sampling processes, preparing and detecting low abundance genes in single cells that are often lost in the extraction process. The main advantages of the proposed NMNP system are 1) high throughput: processing thousands (~50,000) of single cells simultaneously, 2) single cell analysis: nucleic acid detection at the single cell level, 3) multiplex PCR: carries out nested PCR for several genes in single circulating tumor cells, and 4) automated system: single cell manipulation and detection of the PCR results on a chip. The focus of the Phase I work is to design and fabricate a high-throughput nanodrop chip, integrate the NMNP Chip with fluidics and an optical detection system, develop the NMNP multiplex PCR protocol and bioassay, characterize the chip, and evaluate NMNP performance. PUBLIC HEALTH RELEVANCE: Maxwell Sensors Inc. proposes to develop a two step Multiplex Nested PCR system using single circulating tumor cells for cancer diagnosis. This system combines nanotechnology, fluid dynamics, real-time Multiplex PCR, single-cell assays, and circulating tumor cell cancer diagnostics. These assays also have the potential to guide treatment selection based on the molecular profile of the tumor before therapy. Detection of abnormal genes in the intracellular materials provides important clues for early disease diagnosis.

描述（由申请人提供）：使用多重巢式 PCR 系统对白血病进行单细胞诊断。为了更好地对癌症（例如白血病）进行靶向诊断和治疗，需要一种简单的基于血液的检测，该检测可以一致地提供表达信息，同时最大限度地减少患者的不适。目前慢性粒细胞白血病 (CML) 检测方法基于全血细胞计数，显示所有类型的粒细胞都有所增加。用于筛查费城染色体的骨髓活检通常作为 CML 评估的一部分进行，但仅凭骨髓形态不足以诊断 CML。为了选择最佳治疗方法并监测患者的反应，了解患者的白血病类型是否为 CML 非常重要。为了确认诊断，临床医生需要依靠多重 qRT-PCR 检测，使用已知在不同类型白血病中表达差异的基因。然而，目前所有的检测方法都费力且耗时，并且不存在高通量方法来筛选血液样本中存在大量细胞。因此，Maxwell Sensors Inc. 提议使用基于纳米液滴的多重巢式 PCR (NMNP) 系统开发白血病的单细胞诊断，该系统具有处理数千个单个细胞的能力；基于多重 PCR 结果检测循环肿瘤细胞；并分析慢性粒细胞白血病 (CML) 和费城染色体阳性急性淋巴细胞白血病 (Ph+ ALL) 是否存在嵌合 BCR/ABL mRNA。 NMNP 系统自动化所有采样过程，制备和检测单细胞中经常在提取过程中丢失的低丰度基因。所提出的 NMNP 系统的主要优点是 1) 高通量：同时处理数千个 (~50,000) 个单细胞，2) 单细胞分析：单细胞水平的核酸检测，3) 多重 PCR：进行巢式 PCR单个循环肿瘤细胞中的多个基因，以及 4) 自动化系统：单细胞操作和芯片上 PCR 结果的检测。第一阶段工作的重点是设计和制造高通量纳米滴芯片，将 NMNP 芯片与流体和光学检测系统集成，开发 NMNP 多重 PCR 协议和生物测定，表征芯片并评估 NMNP 性能。公共健康相关性：Maxwell Sensors Inc. 提议开发一种两步多重巢式 PCR 系统，使用单个循环肿瘤细胞进行癌症诊断。该系统结合了纳米技术、流体动力学、实时多重 PCR、单细胞分析和循环肿瘤细胞癌症诊断。这些测定还有可能根据治疗前肿瘤的分子谱来指导治疗选择。细胞内物质中异常基因的检测为早期疾病诊断提供了重要线索。