Scott O Sundberg, Carl T Wittwer, Chao Gao, Bruce K Gale
An inexpensive plastic disk disposable was designed for digital polymerase chain reaction (PCR) applications with a microfluidic architecture that passively compartmentalizes a sample into 1000 nanoliter-sized wells by centrifugation. Well volumes of 33 nL were attained with a 16% volume coefficient of variation (CV). A rapid air thermocycler with aggregate real-time fluorescence detection was used, achieving PCR cycle times of 33 s and 94% PCR efficiency, with a melting curve to validate product specificity. A CCD camera acquired a fluorescent image of the disk following PCR, and the well intensity frequency distribution and Poisson distribution statistics were used to count the positive wells on the disk to determine the number of template molecules amplified. A 300 bp plasmid DNA product was amplified within the disk and analyzed in 50 min with 58-1000 wells containing plasmid template. Target concentrations measured by the spinning disk platform were 3 times less than that predicted by absorbance measurements. The spinning disk platform reduces disposable cost, instrument complexity, and thermocycling time compared to other current digital PCR platforms.
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