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

尽管用于生物分析的微流控技术已经取得了相当大的进展，包括液滴微流控技术，它可以将流体扩展到微小的隔间中进行单细胞分析，但将该技术转化为实际应用仍然受到以下因素的阻碍：1)宏观-微观界面不足，无法处理含有复杂基质的大体积生物样品；2)复杂的pcr后处理，用于多重、大规模的基因型分析；3)系统集成进展不足。在这个项目中，我们计划开发一个完全集成的微总量分析系统（�&#56861；TAS），用于在单细胞分辨率下从混合群体中进行高通量大规模微生物检测和基因型鉴定。我们的新平台将包含一个微流体液滴发生器，将单个细菌细胞封装成大量的pl大小的液滴，在那里细胞被裂解和酶反应放置，以促进高通量单细胞PCR扩增。对于大规模的细菌检测和物种鉴定，我们将采用先前验证的16S rRNA PCR检测，结合高分辨率熔融分析（HRMA）进行扩增子序列分析。该平台不仅可以提供高度精确的细菌负荷定量，而且在单细胞水平上结合HRMA可以进行大规模的物种鉴定，以及测量混合群体中存在的细菌物种相对丰度的前所未有的分辨率。

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.