Escherichia coli O157:H7(E.coli)infection has become a global public health and food safety problem.Droplet digital PCR(ddPCR)technology is an emulsion-based endpoint PCR method for independent DNA quantification.Compared to conventional culture method,ddPCR approach represents a new quantitative assay for sensitive and specific detection of nucleic acid.In this study,we use ddPCR method to quantifying copy number of E.coli in tap water.Standard TaqMan primer-probe chemistry was used for ddPCR amplification reactions.Forward and reverse primers and MGB probe with VIC fluorophore was designed according to the sequence of target molecule.For generation of water-in-oil droplet,about 10μL reaction mixture containing 480 Master Mix,primer and probe and 50μL droplet generation oil were aspirated into the chip chamber under negative pressure.The chip was placed on Eppendorf thermocycler with an initial denaturation step(95℃,10 min),followed by 35 cycles of 94℃ for 15s and 60℃ for 30s.Following Poisson statistics with high dilutions of DNA template,each droplet is independently interrogated for the presence of a nucleic acid at single molecule sensitivity.The concentration of the primers and the probe was optimized to enhance the dPCR reaction efficiency and thus the sensitivity.The signals were collected using fluorescence microscope equipped with charge-coupled device(CCD).According to the average size of ~80μm and the number of positive droplet and total droplet,we can calculate the copy number of E.coli DNA.A linear ddPCR response to E.coli DNA concentration was obtained from 0.5% through to 50.0% saturation in a 20,000 droplet assay corresponding to more than 3 orders of magnitude of target DNA copynumber per ddPCR.Next,we will further focus on the pretreatment,i.e.enrichment of E.coli,and the duplex or triplex droplet dPCR system for multiple pathogenic bacterials detection in one assay.