| Autor: |
Ho M; Department of Chemistry, University of Toronto, 80. St. George Street, Toronto, Ontario, M5S 3H6, Canada. aaron.wheeler@utoronto.ca.; Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario, M5S 3E1, Canada., Sathishkumar N; Department of Chemistry, University of Toronto, 80. St. George Street, Toronto, Ontario, M5S 3H6, Canada. aaron.wheeler@utoronto.ca.; Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario, M5S 3E1, Canada., Sklavounos AA; Department of Chemistry, University of Toronto, 80. St. George Street, Toronto, Ontario, M5S 3H6, Canada. aaron.wheeler@utoronto.ca.; Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario, M5S 3E1, Canada., Sun J; Department of Chemistry, University of Toronto, 80. St. George Street, Toronto, Ontario, M5S 3H6, Canada. aaron.wheeler@utoronto.ca., Yang I; Department of Chemical Engineering & Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5, Canada., Nichols KP; Global Health Labs, Bellevue, WA, 98007, USA., Wheeler AR; Department of Chemistry, University of Toronto, 80. St. George Street, Toronto, Ontario, M5S 3H6, Canada. aaron.wheeler@utoronto.ca.; Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario, M5S 3E1, Canada.; Institute of Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario, M5S 3G9, Canada. |
| Abstrakt: |
There is great enthusiasm for using loop-mediated isothermal amplification (LAMP) in point-of-care nucleic acid amplification tests (POC NAATs), as an alternative to PCR. While isothermal amplification techniques like LAMP eliminate the need for rapid temperature cycling in a portable format, these systems are still plagued by requirements for dedicated optical detection apparatus for analysis and manual off-chip sample processing. Here, we developed a new microfluidic system for LAMP-based POC NAATs to address these limitations. The new system combines digital microfluidics (DMF) with distance-based detection (DBD) for direct signal readout. This is the first report of the use of (i) LAMP or (ii) DMF with DBD - thus, we describe a number of characterization steps taken to determine optimal combinations of reagents, materials, and processes for reliable operation. For example, DBD was found to be quite sensitive to background signals from low molecular weight LAMP products; thus, a Capto™ adhere bead-based clean-up procedure was developed to isolate the desirable high-molecular-weight products for analysis. The new method was validated by application to detection of SARS-CoV-2 in saliva. The method was able to distinguish between saliva containing no virus, saliva containing a low viral load (10 4 genome copies per mL), and saliva containing a high viral load (10 8 copies per mL), all in an automated system that does not require detection apparatus for analysis. We propose that the combination of DMF with distance-based detection may be a powerful one for implementing a variety of POC NAATs or for other applications in the future. |
