Miniaturized DNA Sequencer for Identification of Microbial Pathogens

用于微生物病原体鉴定的小型化 DNA 测序仪

• 批准号: 8251255
• 负责人: MICHAEL G POLLACK
• 金额: $ 60.08万
• 依托单位: ADVANCED LIQUID LOGIC
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8251255
• 关键词: Academic Medical Centers; Base Pairing; Binding; Biochemical Reaction; Biochemistry; Chemistry; Clinical; Collaborations; Communicable Diseases; Computer software; DNA; DNA Sequence; DNA amplification; Data; Data Analyses; Databases; Development; Devices; Diagnostic; Equipment; Genetic; Goals; Immunoassay; Investments; Laboratories; Left; Length; Life; Link; Methods; Methylation; Microfluidics; Molds; Nature; Nucleotides; Patients; Performance; Pharmacogenetics; Phase; Preparation; Protocols documentation; Reading; Research; Research Personnel; Running; Sampling; Sequence Determination; Solutions; Specialist; Speed; System; Technology; Testing; Time; Yeasts; base; cancer type; clinical application; cost; design; digital; flexibility; improved; instrument; microbial; miniaturize; next generation; pathogen; prototype; success; tool; tool development

DESCRIPTION (provided by applicant): The ultimate goal of this project is to develop an extremely compact and inexpensive DNA sequence for applications in laboratory and clinical settings. The device will implement pyrosequencing on a disposable droplet based "digital microfluidic" cartridge and will be capable of sequencing 10's to 100's of nucleotides at a very low cost per run and at a low initial equipment investment. This platform will also integrate sample preparation, including DNA amplification, with sequencing to provide the integration and ease-of-use demanded of clinicians and other non-specialists who require DNA sequence data. The initial application for this capability will be microbial pathogen identification. In Phase I, a prototype was developed and the microfluidic protocols and biochemical reaction conditions for pyrosequencing were optimized. As a feasibility demonstration 20-40 base pairs within the ITS2 genetic region were sequenced for 13 different molds and yeasts which provided unambiguous identification of each isolate. In Phase II, this success will be built upon by integrating sample preparation steps including DNA concentration, DNA amplification, and template preparation into the system. The integrated system will be evaluated and compared to other commercially available solutions in terms of speed, cost and accuracy of fungal identification. Further optimization of the pyrosequencing biochemistry will be performed to enable longer read lengths on shorter time scales while retaining high accuracy. Finally, a data analysis tool will be developed to automate base calling and database queries. The final product will represent a streamlined approach to fungal species identification, integrating a number of components of the contemporary pathogen identification workflow. PUBLIC HEALTH RELEVANCE: A compact and inexpensive instrument which can automatically identify fungal isolates by sequencing a small region of their DNA will be developed and tested. This product would greatly improve the ability of clinicians to treat infectious disease.

描述（由申请人提供）：该项目的最终目标是开发一种极其紧凑且廉价的 DNA 序列，用于实验室和临床环境中的应用。该设备将在基于一次性液滴的“数字微流体”盒上实施焦磷酸测序，并且能够以非常低的每次运行成本和较低的初始设备投资对数十至数百个核苷酸进行测序。该平台还将包括 DNA 扩增在内的样品制备与测序相结合，以提供临床医生和其他需要 DNA 序列数据的非专家所需的集成和易用性。该功能的最初应用将是微生物病原体识别。在第一阶段，开发了原型并优化了焦磷酸测序的微流控方案和生化反应条件。作为可行性论证，对 13 种不同的霉菌和酵母的 ITS2 遗传区域内的 20-40 个碱基对进行了测序，这为每个分离株提供了明确的识别。在第二阶段，我们将通过将 DNA 浓缩、DNA 扩增和模板制备等样品制备步骤集成到系统中来建立这一成功。该集成系统将在真菌识别的速度、成本和准确性方面进行评估，并与其他商用解决方案进行比较。将进一步优化焦磷酸测序生物化学，以在更短的时间尺度上实现更长的读取长度，同时保持高精度。最后，将开发数据分析工具来自动化碱基调用和数据库查询。最终产品将代表一种简化的真菌物种鉴定方法，整合了当代病原体鉴定工作流程的许多组成部分。 公共卫生相关性：将开发和测试一种紧凑且廉价的仪器，可以通过对一小部分 DNA 进行测序来自动识别真菌分离株。该产品将极大提高临床医生治疗感染性疾病的能力。