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Contamination with Escherichia coli O157∶H7 (E.coli O157∶H7) is a recurring problem in most European countries and the United States.Rapid methods to detect E.coli O157∶H7 are important to identify the source of outbreaks and to assure public safety.Conventional detection methods1,2, such as culture-based methods, are time and reagent-consuming, low sensitive and labor intensive.Herein, we develop an approach to allow the highly sensitive detection of pathogenic bacteria in a quantitative manner from a background of nonpathogenic bacteria.The method is based upon using a agarose droplets microfluidics in order to perform digital PCR in millions of picolitre droplets combined with flow cytometry techniques3.The agarose emulsion droplet microfluidic technology employed agarose with low melting and low gelling temperature, which was coupled with PCR reverse primer using Schiff-base reaction.E.coli is compartmentalized in droplets at a concentration of less than one bacteria per droplet together with two kinds of fluorescein-labeled forward primers, one specific for the E.coli O157∶H7 and the other for the E.coli K12, which generate red and green fluorescent signals respectively.After PCR, the droplets are then cooled and converted to microbeads carrying the amplified products.The ratio of E.coli O157∶H7 to E.coli K12 is determined by counting the ratio of red to green beads with flow cytometric analysis.The method is, both sensitive and quantitative, the sensitivity being limited only by the number of droplets analyzed.Moreover, the agarose droplets microfluidics will find potential applications in mutant gene detection, gene sequencing, and in the early diagnosis of cancer disease.