Microfluidic Encapsulation of DNAs in Liquid Beads for Digital Loop-Mediated Isothermal Amplification

Digital nucleic acid analysis has emerged as a prominent tool for the detection and absolute quantification of diverse pathogens. Digital loop-mediated isothermal amplification (dLAMP) offers highly sensitive, specific, time-efficient, and cost-effective nucleic acid amplification. However, existing dLAMP techniques face challenges such as droplet merging, reliance on surfactants, restricted partition capacities, and the potential for sample loss during heating. Herein, these issues are addressed by introducing liquid beads for sample partitioning. Compared to microwells, our approach overcomes the limitations of chamber dimensions, enabling the analysis of an unlimited number of digitized targets. Furthermore, our novel approach effectively addresses sample loss and merging during thermal processing and eliminates the need for surfactants. Accurate and reproducible the quantitative detection of the gene cluster XALB1 of leaf scald disease is conducted using dLAMP based on liquid beads to verify its availability. The results demonstrate a high correlation between target concentration and positive signals, indicating the robust performance of our technique. A comparative analysis is then performed between dLAMP using liquid beads and using single droplets. Benchmarking these two techniques highlights the effectiveness of our innovative technique in overcoming existing challenges in dLAMP.