title: Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.
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.
authors.affiliation: Department of Biomedical Engineering, Boston University, Boston, MA, USA.
authors.forename: Shichu
authors.initials: S
authors.lastname: Huang
authors.forename: Jaephil
authors.initials: J
authors.lastname: Do
authors.forename: Madhumita
authors.initials: M
authors.lastname: Mahalanabis
authors.forename: Andy
authors.initials: A
authors.lastname: Fan
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.forename: Lisa
authors.initials: L
authors.lastname: Jepeal
authors.forename: Satish K
authors.initials: SK
authors.lastname: Singh
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Klapperich
mesh_terms: D016360:Clostridioides difficile / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D004247:DNA / Q000235:genetics*; D003967:Diarrhea / Q000382:microbiology*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D013696:Temperature
chemical_list: D004247:DNA
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