Rapid and Sensitive Human-Specific DNA Quantitation Using a Microfluidic Amplification Module at the Point-of-Care.

Abstract

A rapid microfluidic human-specific DNA quantitation assay module was developed for chip-based amplification of the human TH01 and Alu loci in the presence of PicoGreen. The method makes use of the thermal cycler and 488 nm Solid State laser-based optical train that are components of the fully-integrated, sample-in to results out, ANDE Rapid Nucleic Acid Analysis system. The assay was effective in quantitating human DNA from a variety of sample types, including blood, buccal, and forensic touch samples mixed with varying amounts of non-human DNA. The 28-cycle TH01 and 10-cycle Alu reactions were completed in 18 minutes and 7 minutes, respectively. The observed limit of detection (LOD) of the assay is approximately 0.3 ng, and the flexibility of assay design allows an LOD of as little as 0.005 femtograms.Clinical Relevance-We have developed a fully-integrated, sample-in to results-out, Rapid Nucleic Acid Analysis system that characterizes nucleic acid fragments (whether generated by PCR, rt-PCR, sequencing, or SNP reactions) by electrophoresis in plastic microfluidic channels. Here we describe the development, characterization, and validation of the microfluidic quantitation module. The quantitation module is the first that can be incorporated into integrated microfluidic workflows for the analysis of highly-multiplexed clinical diagnostic assays interrogating hundreds of genomic targets in a single sample. In particular, the use of a microfluidic quantitation module allows reaction volumes, thermal cycling conditions, and electrophoretic injection protocols to be determined based on nucleic acid content during and throughout fully-automated processing-dramatically enhancing the power of the fully-automated diagnostic system.