Fig. 3: SMOLT multiplex detection.

a Strategy for detection of multiple targets in a single reaction and single capillary. Each target is detected with a Long Probe of a different length. Beads tethered by a Long-Probe move a distance that is correlated to the Long-Probe length and can be differentiated from beads tethered with probes of a different length based on displacement. b Demonstration of SMOLT multiplexing by detecting rRNA targets using nine Long-Probes of different lengths that generate nine unique peaks in the histogram of bead displacement. Peaks 1–9 correspond to Long-Probes with the following lengths: 3.1, 5.2, 7.4, 9.4, 11.4, 13.4, 15.4, 17.4, and 19.4 kb. c–f Histograms of bead displacement, including only disrupted beads, for multiplex detection of Candida spiked into human whole blood. Three probe pairs simultaneously detected three target sequences. A representative histogram of three independent experiments is shown. c Blank sample, d 30 CFU mL⁻¹ C. albicans only, e 30 CFU mL⁻¹ C. glabrata only, and f 30 CFU mL⁻¹ C. parapsilosis only. Pan-F pan-fungal probe pair peak (green window); Cg C. glabrata probe pair peak (blue window); Ca C. albicans probe pair peak (gray window). The length of the Pan-F, Cg, and Ca Long Probes are 5.4, 7.4, and 9.4 kb, respectively. As expected, C. albicans generated the pan-fungal peak and C. albicans-specific peak. Similarly, C. glabrata generated the pan-fungal peak and C. glabrata-specific peak. In contrast, C. parapsilosis only generated the pan-fungal peak. g Signal generated from multiplex detection versus singleplex detection of either blank, 30 CFU mL⁻¹ or 86 CFU mL⁻¹ of C. parapsilosis. Analysis using unpaired two-tailed Student’s t-test showed that signal generated from multiplex and singleplex detection are indistinguishable for each comparison with p value > 0.1 (n.s. not significant).