Integrated Micro-fluidic Chip Digital Isothermal Amplification Analysis For Rapid Diagnosis Of Escherichia Coli Urinary Tract Infections

Objective:To develop a fully integrated microfluidic assay with digital isothermal amplification for the rapid diagnosis of E.coli caused urinary tract infections.Methods:We designed and fabricated a microfluidic chip integrates nucleic acid extraction,droplet-based sample dispersion and digital isothermal amplification.Nucleic acid extraction on the microchip combines the use of droplet and magnetic bead technologies.The magnetic beads were magnetically transported along a series of microwells containing sample lysis/binding buffer,washing buffer and elution buffer,and then the nucleic acid elution was mixed with the loop-mediated isothermal amplification(LAMP)reaction solution;then,the LAMP mixture was dispersed into droplets by negative pressure-driven fluid shearing,which were collected in the reaction chamber;finally,nucleic acid in the droplets was amplified under isothermal conditions at 65?.After the reaction,the proportion of positive droplets was determined by fluorescence imaging,and the number of targets in a droplet was predicted by fitting the proportion to the Poisson algorithm.We first optimized the analysis conditions by using the standard E.coli sample;and then used batched E.coli clinical samples to verify the feasibility of the analysis method for clinical practice.Results:The integrated microfluidic chip digital isothermal amplification analysis method developed in this research can complete the qualitative and quantitative detection of pathogenic bacteria in a"sample-in-out"manner.Under optimized experimental conditions,the entire analysis process can be completed in 2.5 hours.The detection dynamic range of this method spans 4 orders of magnitude,and the quantitative deviation is<34.75%in the range of 2.36í10~4-1.71í10~7 CFU/mL(R~2=0.9948).The analysis results of batch clinical samples show that this method is in good agreement with the existing bacterial detection methods(Kappa=1,p<0.001).Conclusion:We developed an integrated microfluidic chip digital isothermal amplification method in this work,and demonstrated the feasibility of the method for rapid diagnosis of E.coli urinary tract infections.The results show that this microfluidic chip analysis method has the advantage of simplicity and short analysis time,which is expected to be a powerful tool for rapid pathogen detection in resource-limited conditions.