Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis

PLoS One. 2013;8(3):e60059. doi: 10.1371/journal.pone.0060059. Epub 2013 Mar 28.

Abstract

In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2) pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Clostridioides difficile / genetics
  • Clostridioides difficile / isolation & purification
  • Clostridioides difficile / pathogenicity
  • DNA / genetics*
  • Diarrhea / microbiology*
  • Humans
  • Nucleic Acid Amplification Techniques / methods*
  • Temperature

Substances

  • DNA