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The number of cases involving produce-associated illnesses has been increasing recently, especially those related to pathogen-contaminated irrigation water. Clearly, real-time and extremely sensitive detection of these pathogens is needed to ensure that produce-related farming procedures are safe. In our study, we demonstrated that the use of a microfluidic system can detect Escherichia coli in a water pipe at laminar and turbulent flow regimes. A one-step latex immunoagglutination assay was performed within a microfluidic device that uses fiber optics to detect pathogens. The results were then successfully validated by using cultured E. coli and a salt tracer. The detection of the E. coli was thus accomplished in real time (<5 min per each assay) and at concentrations less than 10 cfu mL-1, suggesting that the system is appropriate for monitoring waterborne pathogens. Specifically, our study found that, in a straight pipe, cell fragments and free antigens of E. coli behave in ways similar to the salt tracer, while viable E. coli cells do not. The computational fluid dynamics model successfully predicted flow dispersion and presents the possibility of modeling the behavior of waterborne pathogens. This study also suggests the possibility of early detection of systemic contamination and timely public health risk assessment before a costly disease outbreak occurs.