Accurate quantification of DNA using on-site PCR (osPCR) by characterizing DNA amplification at single-molecule resolution

Despite the need in various applications, accurate quantification of nucleic acids still remains a challenge. The widely-used qPCR has reduced accuracy at ultralow template concentration and is susceptible to nonspecific amplifications. The more recently developed dPCR is costly and cannot handle high-concentration samples. We combine the strengths of qPCR and dPCR by performing PCR in silicon-based microfluidic chips and demonstrate high quantification accuracy in a large concentration range. Importantly, at low template concentration, we observe on-site PCR (osPCR), where only certain sites of the channel show amplification. The sites have almost identical ct values, showing osPCR is a quasi-single molecule phenomenon. Using osPCR, we can measure both the ct values and the absolute concentration of templates in the same reaction. Additionally, osPCR enables identification of each template molecule, allowing removal of nonspecific amplification during quantification and greatly improving quantification accuracy. We develop sectioning algorithm that improves the signal amplitude and demonstrate improved detection of COVID in patient samples.

Automated summary

Despite the challenges in accurate nucleic acid quantification, we have developed a method that combines the strengths of qPCR and dPCR using silicon-based microfluidic chips. This method demonstrates high quantification accuracy across a wide concentration range and enables the identification of individual template molecules. With this approach, we can remove nonspecific amplification and greatly improve quantification accuracy, as shown in our improved detection of COVID in patient samples using a sectioning algorithm.