Abstract
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 because of 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 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%, whereas 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 et al., 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.
Highlights
Tuberculosis (TB) remains a global challenge with an estimated 10 million infections and 1.3 million deaths per annum worldwide.[1]
The high-gradient magnetic separation (HGMS)-enabled extraction method recovered a total of 10.2 ± 4.03% of spiked DNA, the commercially available Qiagen DNeasy Blood and Tissue kit recovered 17.3 ± 4.65%, and the Chargeswitch gDNA Mini kit recovered 10.1 ± 1.12% (Figure 3)
Extractions were more efficient for urine samples, with the HGMS-enabled extraction yielding 91.2 ± 7.46% of spiked DNA, the commercial Qiagen QIAamp Viral RNA Mini kit recovering 96.5 ± 10.46% of spiked DNA, and the commercial Chargeswitch gDNA Mini kit recovering 69.5 ± 15.4% (Figure 3)
Summary
Tuberculosis (TB) remains a global challenge with an estimated 10 million infections and 1.3 million deaths per annum worldwide.[1] Diagnosis of active, transmissible infections remains a significant public health challenge, in low-resource settings.[2,3] Sputum is the standard patient sample used in both traditional microscopic inspection[2] and nucleic acid-based tests such as GeneXpert.[4] some patient populations, including children and HIV-positive individuals,[5] have difficulty producing sputum. The highly dilute number of targeted biomarkers available for detection and the presence of inhibitors of downstream detection methods limit its utility in nucleic acidbased testing. One fundamental limitation to improved diagnostic sensitivity remains the development of sample extraction and concentration methods, which convert dilute biomarkers into pure, inhibitor-free samples appropriate for downstream detection
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