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摘要
Digital nucleic acid analysis is a favorable tool for rapid molecular detection of pathogens due to its absolute quantification capability without the necessity of standard curves. However,current digital nucleic acid analysis platforms commonly perform the workflow with discrete facilities,which complicates the operation process and lengthens the turnaround time,therefore restricting its adoption in resource-limited settings. To address this issue,an integrated microfluidic droplet digital isothermal amplification system was developed in this work. This system integrated sequential droplets-based nucleic acid extraction unit,syringe-vacuum actuated droplet generation unit,and droplet digital loop-mediated isothermal amplification (LAMP)unit, achieving digital analysis of bacterial nucleic acid within 1.5 h in an integrated manner. The nucleic acid extraction unit could complete E. coli genomic DNA extraction with the efficiency of 93.68% & PLUSMN;32.38%,the droplet generation unit could produce 20000 droplets in 4 min with the relative standard deviation (RSD)less than 10%. The LAMP reaction could be performed with a linear dynamic range of 4 orders of magnitude (2.36 x 104 - 1.71 x 1 07 CFU/ mL). Compared to traditional culture method,the results obtained from analysis of E. coli in UTI clinical samples (n =13)showed that both the detection sensitivity and specificity of this digital nucleic acid analysis method were 100% (Kappa= 1,p < 0.01). With the advantages of accurate quantification and ease of operation,this integrated microfluidic droplet digital isothermal amplification system was expected to be a favorable tool for rapid point-of-care detection of pathogens in resource-limited settings.
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