Nucleic acid-based diagnostic tests often require isolation and concentration of nucleic acids from biological samples. Commercial purification kits are difficult to use in low-resource settings due to their cost and insufficient laboratory infrastructure. Several recent approaches based on the use of magnetic beads offer a potential solution, but remain limited to small volume samples. We have developed a simple and low-cost nucleic acid extraction method suitable for isolation and concentration of nucleic acids from small or large sample volumes. The method uses magnetic beads, a transfer pipette, steel wool, and an external magnet to implement high-gradient magnetic separation (HGMS) to retain nucleic acid-magnetic bead complexes within the device’s steel wool matrix for subsequent processing steps. We demonstrate the method’s utility by extracting tuberculosis (TB) DNA from both sputum and urine – two typical large volume sample matrices (5-200 mL) using guanidine-based extraction chemistry. Our HGMS-enabled extraction method is statistically indistinguishable from commercial extraction kits when detecting a spiked 123-base DNA sequence. For our HGMS-enabled extraction method, we obtained extraction efficiencies for sputum and urine of approximately 10% and 90%, while commercial kits obtained 10-17% and 70-96% respectively. We also used this method previously in a blinded sample preparation comparison study published by Beall , 2019. Our manual extraction method is insensitive to high flow rates and sample viscosity, with capture of ~100% for flow rates up to 45 mL/min and viscosities up to 55 cP, possibly making it suitable for a wide variety of sample volumes and types and point-of-care users. This HGMS-enabled extraction method provides a robust instrument-free method for magnetic bead-based nucleic acid extraction, potentially suitable for field implementation of nucleic acid testing.
March 10, 2020
February 18, 2020
October 3, 2017
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