Digital Quantification of Human Viral RNA and DNA Using a Self-Digitization Chip

Abstract

Digital nucleic acid quantitation methods show excellent sensitivity and specificity for pathogen detection. Droplet digital PCR (ddPCR) is the most advanced digital nucleic acid quantitation method and has been commercialized, but is not suitable for many point-of-care applications due to its complex instrumentation. Here we describe a simple microfluidics-based self-digitization (SD) chip for quantifying nucleic acids at the point of care with minimal instrumentation. We demonstrate the clinical diagnostic capability of this platform by applying it to quantifying human viral DNA and RNA. SD chips with a range of well numbers and volumes are tested, and isothermal methods are used to amplify the DNA and RNA to a detectable level. Sample concentration is determined based on the measured volume in the wells and the number of wells with fluorescence greater than a threshold based on a Poisson distribution. Concentration measurements over the low concentration range of 0-100 molecules/μL showed a strong correlation (R² = 0.99) with measurements using a real-time PCR assay, demonstrating the sensitivity and specificity of the SD chip platform.

Keywords: Digital nucleic acid quantification; Isothermal amplification; Point-of-care (POC) tests; Self-digitization (SD) chip; Viral RNA and DNA.