B-228 Partition-free Digital Polymerase Chain Reaction: A Novel Approach to Enable Rapid Turnaround Time With Digital Accuracy

Abstract

Abstract Background Partition-free digital PCR (dPCR) is a new approach developed to provide absolute quantification in less than 10 min. Partition-free eliminates the need for droplets or physical wells that need to be filled and sealed. The technique relies on rapid thermal cycling and microfluidics to limit diffusion and enable the identification of individual amplicon clusters originating from a single target nucleotide template. Rapid heating and cooling were achieved with a thermal cycling engine comprised of a broadband LED with a sapphire block that is both a light guide and heatsink. Sample is distributed through a custom microfluidic cartridge containing 64 microchannels for optical quantification. Methods The feasibility of partition-free dPCR was tested with primers targeting MTHFR c.665. A titration was performed on three 4X serial dilutions of cell line genomic DNA in triplicate. Fluorescent profiles were generated for all 64 microchannels from an image captured after 35 PCR cycles, where amplicon clusters were identified. Fluorescent peaks of the amplicon clusters were counted and quantified via a custom analysis algorithm, which uses Poisson statistics to calculate concentration. Results The quantification results obtained from the partition-free dPCR system were compared to concentration measurements taken via a Bio-Rad QX200 platform. Concentrations of 69.4 ± 0.8, 15.4 ± 1.5, and 3.3 ± 0.6 copies/µL (R2 = 0.9997) were measured on the partition-free system, and concentrations of 67.8 ± 3.6, 17.2 ± 1.1, and 2.8 ± 0.2 copies/µL (R2 = 0.9995) were measured on the Bio-Rad QX200. Conclusion It has been demonstrated that absolute quantification can be achieved in less than 10 min with a partition-free dPCR platform, and shows results comparable to a QX200 droplet dPCR system. The partition-free rapid dPCR technology described herein allows for the exploration of new avenues in molecular diagnostics, with the potential to bring absolute quantification of dPCR to a Point-of-Care setting.