A Magnetic Bead-Based Method for Concentrating DNA from Human Urine for Downstream Detection

Abstract

Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×103 to 5×108 copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×106, 14×106, and 8×106 copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR.

A Prototype Biomarker Detector Combining Biomarker Extraction and Fixed Temperature PCR

“…6 A 67 bp amplicon of the IS6110 sequence was amplified using 0.2 µM forward primer (5′ ACC AGC ACC TAA CCG GCT GTG G 3′) and 0.2 µM reverse primer (5′ CAT CGT GGA AGC GAC CCG CCA G 3′). 12 Chamber 1 ( Fig. 1) consisted of 100 µL TE buffer, 5 µL IS6110 DNA at a concentration of 10 6 copies/µL, 300 µL binding buffer (4M guanidine thiocyanate, 25 mM sodium citrate pH 7.0), and 20 µL Dynabeads MyOne Silane magnetic beads (~6 × 10 8 beads; Life Technologies 37002D, Carlsbad, CA).…”

A Rapid and Automated Device for Purifying Nucleic Acids

“…The usefulness of magnetic beads in the microfluidics has already been reported in the bioassay field. [9][10][11][12][13] Magnetic beads are easily controlled from the outside of the camber, and in microfluidics, they are indirectly transferred between several reagents for lysing, extracting, and washing. We also used a combination of superparamagnetic beads and magnetic force for purifying nucleic acids.…”

Development of a Novel and Rapid Fully Automated Genetic Testing System

“…9 Several bioassays have also already demonstrated the utility of magnetic beads in microfluidics. [10][11][12][13] Connection of such a cartridge extraction and purification system 9 to our novel droplet-real-time-PCR system 14 allows PCR tests to be performed simply in high-throughput assays by simple pouring of the sample into the cartridge. This system can eliminate the problems associated with delivering materials such as nucleic acids, proteins, and cells that may adhere to sample tubes and allows liquid handling of all processes.…”