Ultraspecific Multiplexed Detection of Low-Abundance Single-Nucleotide Variants by Combining a Masking Tactic with Fluorescent Nanoparticle Counting.

To be able to detect simultaneously multiple single-nucleotide variants (SNVs) with both ultrahigh specificity and low-abundance sensitivity is of pivotal importance for molecular diagnostics and biological research. In this contribution, we for the first time developed a multiplex SNV detection method that combines the masking tactic with fluorescent nanoparticle (FNP) counting based on the sandwich design. The method presents a rivaling performance due to its advantageous features: the masking reagent was designed to hybridize with an extremely large amount of the wild-type sequence to render the assay with high specificity; FNP counting provides a sensitive multiplexed SNV detection; the sandwich design facilitates an easy separation to make the detection free of interferences from the matrix. For single SNV target discrimination, including the 6 most frequently occurring DNA KRAS gene mutations and 2 possible RNA KRAS gene mutations as well as 11 artificial mutations, the discrimination factor ranged from 204 to 1177 with the median being 545. Among the tested 19 SNVs, abundances as low as 0.05% were successfully identified in 14 cases, and an abundance as low as 0.1% was identified for the remaining 5 cases. For multiplexed detection of SNVs in the KRAS gene, abundances as low as 0.05-0.1% were achieved for multiple SNVs occurring at the same and different codons. As low as 0.05% low-abundance detection sensitivity was also achieved for PCR amplicons of human genomic DNA extracted from cell samples. This proposed method presents the potential for ultrahigh specific multiplexed detection of SNVs with low-abundance detection capability, which may be applied to practical applications.