Integrated microfluidic device of DNA extraction, recombinase polymerase amplification and micro-capillary electrophoresis for sample-to-answer detection of Salmonella Typhimurium

Abstract

Foodborne pathogens pose a significant global health threat, leading to millions of illnesses and deaths annually. Our study introduces an integrated microfluidic chip for rapid molecular diagnostics of foodborne pathogens. The proposed device can perform DNA extraction, recombinase polymerase amplification (RPA), and micro-capillary electrophoresis (µCE) in a sequential manner. With a compact copper-based thermal unit for RPA and precise on-off power supply for the µCE, we could achieve swift detection InvA gene of Salmonella Typhimurium within 75 min. The entire process includes 10 min for sample lysis, 10 min for genomic DNA extraction, 50 min for the RPA reaction, and 5 min for µCE operation and peak assignment. Within 5 min, we were able to detect the presence of the InvA gene of S. Typhimurium (161 bp) with a LOD of 10³ CFU/mL in both bacterial culture and spiked milk samples. We used a short bracket ladder (121 bp) and a long bracket ladder (203 bp) to accurately assign the target peak in the electropherogram, and the relative migration time of target gene over two bracket ladders was 0.449 ± 0.0094. Notably, our platform is compact, with all components neatly arranged within a 3D printed frame measuring 12 cm (width) × 14 cm (length) × 2.7 cm (height), excluding the in-house detector. This integrated microsystem from the sample injection to the CE analysis presents promising prospects for advanced on-site pathogen detection, and lays the groundwork for future molecular diagnostic platform to combat foodborne illnesses and understanding of pathogen genome and variations.