Miniaturized DNA Sequencer for Identification of Microbial Pathogens

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和廉价的DNA序列，用于实验室和临床环境。该设备将在基于一次性液滴的“数字微流体”墨盒上实现焦磷酸测序，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10到100个核苷酸进行测序。该平台还将整合样品制备（包括DNA扩增）和测序，为需要DNA序列数据的临床医生和其他非专业人员提供所需的集成和易用性。这种能力的最初应用将是微生物病原体鉴定。在第一阶段，开发了原型，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ITS2遗传区域内的20-40个碱基对进行了测序，并对每个分离物进行了明确的鉴定。在第二阶段，这一成功将建立在整合样品制备步骤，包括DNA浓度，DNA扩增和模板制备到系统中。将对集成系统进行评估，并在真菌鉴定的速度、成本和准确性方面与其他市售解决方案进行比较。将进一步优化焦磷酸测序生物化学，以实现在更短的时间尺度上更长的读取长度，同时保持较高的准确性。最后，将开发一个数据分析工具，使基地呼叫和数据库查询自动化。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。

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.