Fig. 4: Multiplexed detection of SARS-CoV-2 RNA fragments.

a Genome map showing the full length with annotated regions of SARS-CoV-2. Position of sequences in the ORF1b, S and N genes that have been chosen for detection are marked in magenta. b (i) Schematic representations of a 3-site DNA molecular probe (9.1 kbp) and the binding to ORF1b, S, and N gene targets, respectively. The probe for an individual target is assigned to a specific position along the dsDNA and bound to the first half of the target RNA sequence. A biotinylated sequence is used as a signal enhancement probe to bind the second half of the target sequence, and streptavidin is used to enhance the signal. Sequences for the selected ORF1b, S, and N gene targets are shown, and the binding segment of the dsDNA carrier and biotinylated probe are highlighted in orange and blue, respectively. (ii) Representative events are shown for the individual genome target binding with secondary peaks and are observed at the respective position. (iii) histogram of the normalised frequency of the fractional secondary peak position for each binding genome fragment (n = 100). The detailed secondary peak information is shown in Supplementary Fig. S21. c Schematics for the 3-site molecular probe binding to two (i-iii) or three (iv) target gene fragments and the resulting translocation events are shown in the bottom panel. d Statistics for simultaneous detection of the three RNA targets range from 0.2 pM to 2 nM. e Calibration curve for the N gene binding ratio and the target RNA concentration. All the translocation experiments were performed with 200 pM of molecular probe in 2 M LiCl buffer (5 mM MgCl₂, 10 mM Tris–HCl, 1 mM EDTA, pH = 8) at an applied potential bias of 300 mV. Error bars in e represent the standard deviation of three independent experimental repeats and the measure of the centre represents their corresponding mean value. Source data are provided as a Source Data file.