Microfluidic Single-Cell Melting Curve Analysis for Broad-Scale Detection of Microbial Organisms

用于大规模检测微生物的微流控单细胞熔解曲线分析

• 批准号: 1159771
• 负责人: Jeff Wang
• 金额: $ 39.79万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1159771&HistoricalAwards=false
• 关键词: Microfluidic; Single; Cell; Melting; Curve

Despite considerable progress made in developing microfluidictechnologies for biological analysis, including droplet microfluidics to enable scalable partitioning of fluidinto tiny compartments for single cell analysis, transforming the technology into practical applications arestill hindered by 1) inadequate macro-to-micro interface to cope with large volume biological samplescontaining complex matrix; 2) complicated post-PCR processing for multiplexed, broad-scale genotypicanalysis; and 3) insufficient progress toward system integration.In this project, we plan to develop a fully integrated micro total analysis system (��TAS) for highthroughput broad-scale microbial detection and genotypic identification from a mixed population at singlecell resolution. Our novel platform will incorporate a microfluidic droplet generator to encapsulate singlebacterial cells into a large quantity of pL-sized droplets where the cells are lysed and enzymatic reactionstake place to facilitate high throughput single cell PCR amplification. For broad-scale bacterial detectionand species identification, we will incorporate a previously validated 16S rRNA PCR assay coupled withhigh resolution melt analysis (HRMA) for amplicon sequence analysis. This platform will not only providehighly accurate quantification of bacterial load, but the incorporation of HRMA at the single cell levelpermits both broad-scale species identification, as well as unprecedented resolution in measuring relativeabundance of bacterial species present in a mixed population.

尽管在开发用于生物分析的微流体技术方面取得了相当大的进展，包括液滴微流体技术，以使得能够将流体可缩放地分配到用于单细胞分析的微小区室中，但是将该技术转化为实际应用仍然受到以下因素的阻碍：1）不足以科普含有复杂基质的大体积生物样品的宏-微界面;在本项目中，我们计划开发一个完全集成的微全分析系统（��TAS），用于高通量大规模微生物检测和混合种群的基因型鉴定。我们的新平台将结合微流体液滴发生器，将单个细菌细胞封装到大量的pL大小的液滴中，在液滴中细胞被裂解，酶促反应发生，以促进高通量单细胞PCR扩增。对于大规模的细菌检测和菌种鉴定，我们将采用先前验证过的16 S rRNA PCR检测，结合高分辨率熔解分析（HRMA）进行扩增子序列分析。该平台不仅提供了细菌载量的高度准确的定量，而且在单细胞水平上掺入HRMA允许大规模的物种鉴定，以及测量混合群体中存在的细菌物种的相对丰度的前所未有的分辨率。