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

Johns Hopkins University, Baltimore MD

Investigators

Abstract

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