Droplet Digital Microfluidic Assay Enables Rapid Detection Of Carbapenem-Resistant Bacteria

Objective: The research is expected to realize qualitative and quantitative analysis of carbapenem-resistant bacteria with the help of droplet digital microfluidic chromogenic assay,providing rapid and accurate diagnostic information for carbapenem-resistant bacteria infectious diseases.Methods: We design and process a microfluidic chip with parallel droplet analysis modules,which is characterized by the rapid realization of large-scale sample dispersion of each modules by using a negative pressure providing syringe.Firstly,the droplet formation conditions were optimized to ensure reliable bacterial dispersion.Then,the qualitative and quantitative effect of carbapenem-resistant bacteria were investigated by using Escherichia coli quality control strain.Finally,a number of clinical samples were tested by this method.Results:The chip structure and droplet chemical composition were optimized to ensure efficient droplet generation and long-term stability.Under the negative pressure drive of the syringe,droplet based bacterial dispersion can be completed within 3 min,and droplet sizes generated by the six parallel units all conform to the normal distribution.The experimental conditions determined by the Escherichia coli quality control strain were as follows: droplet diameter of 30 ?m,resazurin final concentration of 400 ?mol· L-1 and incubation time of 3.5 h.In this method,there was a significant difference in fluorescence intensity between the droplet with and without bacteria(non-parametric rank sum test,p<0.01).The dynamic detection range was 105-108 CFU· m L-1,with CVs< 5%.Its quantitative results are consistent with the plate counting method(R2=0.99225).Under the condition of culture for 3.5 hours,the antibacterial rate of 4?g·m L-1meropenem against carbapenem-sensitive bacteria was more than 85.11%,while that of the carbapenem-resistant bacteria was less than 20.08%,suggesting that this method could effectively identify carbapenem-resistant bacteria.The results of mass clinical samples showed that the consistency between the droplet digital antibiotic sensitivity assessment and the disk diffusion method was 95.83%.Conclusion:The study developed a rapid detection method for carbapenem resistant bacteria based on droplet microfluidic technology and the resazurin chromogenic reaction which can identify the presence of bacteria in samples within only 3.5 hours and conduct accurate quantification,as well as antibiotic sensitivity testing based on accurate quantification.This method has the characteristics of parallelization,integration,miniaturization,simple operation,rapid and accurate quantification,so it is expected to provide a powerful and rapid detection method for carbapenem-resistant bacteria in clinic.