Low-Resource Nucleic Acid Extraction Method Enabled by High-Gradient Magnetic Separation

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 acidmagnetic 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.

An automated, high-throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma

“…3g). Furthermore, adding isopropanol to the LBS (for a final concentration of 50%) has been used to isolate cell-free DNA from sputum and urine (49) to promote DNA precipitation onto the MB (50). When using the Thermo Fisher chemistry, we found that isopropanol decreased recovery of mtDNA and nDNA significantly in plasma (Fig.…”

An automated, high throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma

“…3g). Furthermore, adding isopropanol to the LBS (for a final concentration of 50%) has been used to isolate cell-free DNA from sputum and urine (44) to promote DNA precipitation onto the MB (45). When using the Thermo Fisher chemistry, we found that isopropanol decreased recovery of mtDNA and nDNA significantly in plasma (Fig.…”

Advances in point-of-care nucleic acid extraction technologies for rapid diagnosis of human and plant diseases

“…During the RPA assay, the heated printer bed was used to directly heat the RPA tubes, which simplified the overall complexity of the system by eliminating two different temperature water baths and sample transportation between these baths. For on-site nucleic acid purification from urine samples, Pearlman et al (2020) presented a high-gradient magnetic separation (HGMS) technique to capture DNA binding magnetic particles in the steel wool matrix ( Fig. 7 b).…”

“…(b) Schematic of a high-gradient magnetic separation (HGMS) enabled nucleic acid extraction method (reproduced with permission from Ref. ( Pearlman et al, 2020 ), © American Chemical Society 2020). (c) Schematic illustration of a chromatography paper-based nucleic acid extraction system for Chlamydia trachomatis detection (reproduced with permission from Ref ( Linnes et al, 2014 )., © The Royal Society of Chemistry (2014).…”