Fluorescence-coded logarithmic-dilution digital droplet PCR for ultrawide-dynamic-range nucleic acid quantification

Biosensors and Bioelectronics(2023)

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摘要
Digital PCR (dPCR) is considered the next generation of nucleic acid detection for its ability of absolute quantification and high sensitivity. However, when compared to the current gold standard, quantitative PCR (qPCR), dPCR is falling behind by several orders of magnitude in dynamic range, which limits its clinical applicability. Here we present fluorescence-coded logarithmic-dilution digital droplet PCR (Flodd-PCR) that features a dynamic range across 7 orders of magnitude, over 2 orders higher than conventional dPCR (4–5 log range) and approaching that of qPCR (7–8 log range). Flodd-PCR realizes such a wide dynamic range by dividing ∼20,000 droplets into 4 groups, each featuring a unique dilution factor of the loaded DNA template and thus a shifted dynamic range. This is achieved by a microfluidic chip that performs multi-step serial dilution (20–925 folds) and droplet generation. The post-PCR droplets can be clustered in silico based on their dilution indicator fluorescence and analyzed independently. Experimentally, Flodd-PCR can detect 4–20,000,000 copies/μL (cp./μL) of the synthetic human papillomavirus (HPV) DNA and outperforms standard dPCR when analyzing clinical HPV samples. Furthermore, Flodd-PCR can be implemented with existing dPCR system set-up with minimal adjustment, and therefore will also have wide practicality in different applications which conventional dPCR has already demonstrated.
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