Abstract
There has been an urgent need to quickly screen and isolate patients with viral infections from patients with similar symptoms at point-of-care. In this study, we introduce a new microfluidic method for detection of various viruses using rolling circle amplification (RCA) of pathogens on the surface of thousands of microbeads packed in microchannels. When a targeted pathogen meets the corresponding particular template, the DNAs are rapidly amplified into a specific dumbbell shape through the RCA process, forming a DNA hydrogel and blocking the flow path formed between the beads. Due to the significant increase in reaction surface area, the detection time was shortened to less than 15 min and the detection limit of various pathogens has been reached to 0.1 pM. By injecting the stained liquid, the existence of the target pathogens in a sample fluid can be determined with the naked eye. Furthermore, by integrating multi-channel design, simultaneous phenotyping of various infective pathogens (i.e., Ebola, Middle East respiratory syndrome (MERS), and others) in biological specimens can be performed at a point-of-care.
Highlights
When infectious viruses are spreading, the best solution is to rapidly and accurately detect infectious agents and isolate carriers to prevent further spread (Allegranzi et al, 2011)
If the primers are successfully immobilized on the surface of microbeads, they combine with the complementary fluorescent probe (FAM)
The percentage of beads with a high fluorescence intensity (> 103) was 99.9% for Sepharose beads and 0.1% for PS beads. These results indicate that primers were densely immobilized on the surface of the Sepharose beads but not on that of the PS microbeads
Summary
When infectious viruses are spreading, the best solution is to rapidly and accurately detect infectious agents and isolate carriers to prevent further spread (Allegranzi et al, 2011). Through RCA of complementary targets in microfluidic channels, amplified DNA forms DNA hydrogels by adding a dumbbell-shaped padlock probe (Lee et al, 2015). The long process time is mainly due to the limited surface area for pathogen DNA amplification, which occurs at the microchannel surfaces. It requires a large number of DNA strands to form the hydrogel and to block a wide cross-sectional area of the microchannel. The bead-based RCA processes encountered various difficulties, including inefficient DNA hybridization and enzymatic reactions, due to physical properties of the bead surface (Sato et al, 2013)
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