Bare glassy nanopore for length-resolution reading of PCR amplicons from various pathogenic bacteria and viruses

In this study, it is confirmed that without addition of organic solvent and embedding polymer hydrogel into glass nanopore, bare glass nanopore can faithfully measure various lengths of DNA duplexes from 200 to 3000 base pairs with 200 base pairs resolution, showing well-separated peak amplitudes of blockage currents. Furthermore, motivated by this readout capability of duplex DNA, amplicons from Polymerase Chain Reaction (PCR) amplification are straightforwardly discriminated by bare glassy nanopore without fluorescent labeling. Except simultaneous discrimination of up to 7 different segments of the same lambda genome, various pathogenic bacteria and viruses including SARS-CoV-2 and its mutants in clinical samples can be discriminated at high resolution. Moreover, quantitative measurement of PCR amplicons is obtained with detection range spanning from 0.75 aM to 7.5 pM and detection limit of 7.5 aM, which reveals that bare glass nanopore can faithfully disclose PCR results without any extra labeling.

Automated summary

In this study, it has been confirmed that bare glass nanopores can accurately measure various lengths of DNA duplexes without adding organic solvent and embedding polymer hydrogel. The measurement has a resolution of 200 base pairs and shows well-separated peak amplitudes of blockage currents. By utilizing this capability, amplicons from PCR amplification can be discriminated without fluorescent labeling. This method can also discriminate various pathogenic bacteria and viruses, including SARS-CoV-2 and its mutants, in clinical samples with high resolution. Furthermore, the bare glass nanopore can quantitatively measure PCR amplicons without the need for extra labeling, with a detection range from 0.75 aM to 7.5 pM and a detection limit of 7.5 aM.