Detection of single-based mutation (SbM), which is of ultra-low abundance against wild-type alleles, are typically constrained by the level of multiplexing, sensitivity for single-base resolution and quantification accuracy. In this work, an electrochemical quantitative polymerase chain reaction (E-PCR) platform was developed for multiplexed and quantitative SbM analysis in limited and precious samples with single-nucleotide discrimination. A locked nucleic acid (LNA)-mediated multiplexed PCR system in a single, closed tube setup was firstly constructed to selectively amplify the SbM genes while suppressing the wild-type alleles. The amplicons were detected simultaneously through hybridization with the sequence-specific hairpin probes anchored on a reduced graphene oxide-gold nanoparticles functionalized electrode surface. With the inclusion of an LNA-mediated PCR step upstream of the electrochemical detection, we improved the limit of detection (LOD) by 2 orders of magnitude, down to an ultralow-level of 5 copies μL-1. The platform achieved an ultra-sensitive and specific detection with 0.05% against a background of 10, 000 copies of wild-type alleles. It is highly adaptive and has the potential to enable expanded multiplexed detection in parallel, thus providing a universal tool for multiplexed SbM identification.
Keywords: E-PCR platform; LNA-Mediated PCR; Multiplexed detection; Single-based mutation.
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