(134h) Integrated Point of Care Device for Nucleic Acid Extraction, Isothermal Amplification, and Fluorescence-Based on-Line Detection of Viral Genetic Material

Early diagnosis of viral diseases is vital for accurate treatment and containment of infections, and it leads to better outcomes for the patient and effective protection for the community. Motivated by recent epidemics (i.e., 2014 Ebola West Africa and 2015-16 Zika outbreaks), the World Health Organization has called for rapid, safe, and cost-effective virus diagnostic systems to be used at point-of-care (POC) locations.

Nucleic acid amplification methods have the accuracy, sensibility, and specificity needed to make them the tools of choice for viral diagnostics at early infection stages. However, conventional amplification techniques are poorly suited for POC application. Molecular diagnostic usually consists of three steps: genetic material extraction, amplification, and detection. Typically, the accomplishment of these three steps demands well-trained technical staff and costly reagents and equipment (i.e., an extraction reagent kit, a thermocycler, an electrophoresis chamber, and a trans-illuminator). These requirements increase diagnostic expenses and turnaround time, thereby limiting testing availability, especially in underprivileged or remote areas.

Herein, we present an integrated, three-stage, one-pot strategy to solve these drawbacks. In the first stage, a single and short high-voltage electric pulse releases viral nucleic acid material confined within infected cells, making it accessible for amplification. In the second stage, the genetic material is amplified in the same container by an isothermal amplification method, namely Recombinase Polymerase Amplification (RPA). Finally, the amplification product is detected by addition of GelRed® DNA-dye to the reaction mix, excitation with 360 nm UV light, and photodiode detection of the emitted fluorescence. All these steps are performed in a simple, cost effective, and portable device. Moreover, the on-line quantitative detection of the amplification product can be conducted using a simple Arduino-based module. We have demonstrated the use of this integrated device by quantitative detection of synthetic nucleic acid from Ebola, Zika, and influenza viruses released from bacterial and mammalian cells.

The minimal reagent usage and the lack of any requirement for specialized training, along with the simplicity, portability, and low cost of this device, make it suitable for cost-effective use at POC locations.