Low-Resource Nucleic Acid Extraction Method Enabled by High-Gradient Magnetic Separation
| Autor: | Anna E. Wolfe, Nicholas M. Adams, Patricia K. Russ, Cassandra Wessely, Megan E. Pask, Mindy Leelawong, Kelly A. Richardson, Frederick R. Haselton, Stephanie I. Pearlman |
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| Rok vydání: | 2020 |
| Předmět: |
DNA
Bacterial high-gradient magnetic separation Materials science Low resource Magnetic separation Real-Time Polymerase Chain Reaction 01 natural sciences Specimen Handling Magnetic Bead Separation 03 medical and health sciences Nucleic Acids Humans nucleic acid extraction General Materials Science Sample preparation 030304 developmental biology 0303 health sciences Bacteriological Techniques Chromatography sample preparation 010401 analytical chemistry Sputum Diagnostic test Mycobacterium tuberculosis Isolation (microbiology) magnetic bead separation 0104 chemical sciences qPCR tuberculosis low-resource Nucleic acid Magnets Extraction methods Research Article |
| Zdroj: | ACS Applied Materials & Interfaces |
| ISSN: | 1944-8252 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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