Amplification-Free, Sequencing-Free, Detection of Viral RNAs with Variant Specification by Discrete Nanocounting

Abstract

This study describes an amplification-free, sequencing-free platform (NanoPick-array) for fast analysis of viral RNAs. The platform combines selective short-cut of viral RNAs, cherry-picking isolation of target genes, and micro-arrayed discrete nanoimaging to enable the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at a concentration of 60 copies µL⁻¹, a detection limit that is hardly achieved by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based methods without amplification, regardless of electrochemical or colorimetric approaches. Notably, the NanoPick-array provides specific virus variant information with differentiation to single-nucleotide genetic mutation. The avoidance of the amplification procedure gives direct quantification of viral copy number and reduces false positive results caused by amplicon contamination; the sequencing-free viral variant specification significantly reduces the turn-over time for the acquisition of a complete diagnostic viral picture within just 2 h. In a demonstration using clinical samples with a wide range of viral loads of cycle threshold (Ct) value ranging from 18 to 36, the technique achieves an overall accuracy of 89.7% viral detection and 100% accuracy for identifying all Delta variants. The viral detection accuracy is further tested to be 100% for the clinical samples with Ct values around or less than 28.