Automated nucleic acid extraction from whole blood, B. subtilis, E. coli, and Rift Valley fever virus on a centrifugal microfluidic LabDisk

Abstract

We present total nucleic acid extraction from whole blood, Gram-positive Bacillus subtilis, Gram-negative Escherichia coli, and Rift Valley fever RNA virus on a low-cost, centrifugal microfluidic LabDisk cartridge processed in a light-weight (<2 kg) and portable processing device. Compared to earlier work on disk based centrifugal microfluidics, this includes the following advances: combined lysis and nucleic acid purification on one cartridge and handling of sample volumes as large as 200 μL. The presented system has been validated for logarithmic dilutions of aforementioned bacteria and viruses from various sample matrices including blood plasma and culture media and extraction of human DNA from whole blood. Recovered DNA and RNA concentrations in the eluate were characterized by quantitative PCR to: 58.2–98.5%, 45.3–102.1% and 29.5–34.2% versus a manual reference for Bacillus subtilis, Escherichia coli and Rift Valley fever virus, respectively. For extraction of human DNA from whole blood, similar results for on-disk ((10.1 ± 7.6) × 10⁴ DNA copies) and manual reference extraction ((10.2 ± 6.3) × 10⁴ DNA copies) could be achieved. Eluates from on-disk extraction show slightly increased ethanol concentrations of 4.1 ± 0.3% to 5.5 ± 0.2% compared to a manual reference (2.0 ± 0.5% to 3.6 ± 0.6%). The complete process chain for sample preparation is automatically performed within ∼30 minutes, including ∼15 minutes lysis time. It is amenable to concatenation with downstream modules for multiplex nucleic acid amplification as recently demonstrated for panel testing of various pathogens at the point of care.