Method for Lysis and Paper-based Elution-free DNA Extraction with Colorimetric Isothermal Amplification

Abstract

Abstract Nucleic acid amplification testing has great potential for point-of-need diagnostic testing with high detection sensitivity and specificity. Current sample preparation is limited by a tedious workflow requiring multiple steps, reagents and instrumentation, hampering nucleic acid testing at point of need. In this study, we present then use of mixed cellulose ester (MCE) paper for DNA binding by ionic interaction and fluid transport by wicking. The poly(ethylene) glycol-based (PEG) reagent simultaneously provides the alkalinity effect for alkaline lysis and crowding effects for ionic DNA binding of the DNA under high salt conditions. Using a narrow strip of paper, the freed DNA concentrates at the paper tip, while the wicking removes the sample matrix when briefly washing using 40% isopropanol, a 15 in process that is followed by on-paper amplification after a drying step. Colourimetric loop-mediated isothermal amplification enabled the detection of 102 CFU/mL of Escherichia coli (E. coli) from culture media and the detection of E. coli in milk < 103 CFU/mL (10 CFU) after incubation at 68°C for 60 min, demonstrating applicability of the method to complex biological samples.