Detection of HIV-1 Minority Variants Containing the K103N Drug-Resistance Mutation Using a Simple Method to Amplify RNA Targets (SMART) Kenneth Morabito,

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Detection of HIV-1 Minority Variants Containing the K103N Drug-Resistance Mutation Using a Simple Method to Amplify RNA Targets (SMART) Kenneth Morabito, Rami Kantor, Warren Tai, Leeann Schreier, Anubhav Tripathi The Journal of Molecular Diagnostics Volume 15, Issue 3, Pages 401-412 (May 2013) DOI: 10.1016/j.jmoldx.2013.02.005 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Overview of SMART. 1) RNA sample is isolated from a clinical sample and put into a TE buffer solution. 2) Streptavidin-coated beads conjugated with biotinylated capture oligonucleotides and SMART probes are added to the solution. 3) The solution is added to W1 of the SMART microchip, and a magnet is used to separate the bead-bound complex from the unbound structures. 4) SMART probes are amplified via a modified NASBA scheme. 5) Sample is quantified in real-time using molecular beacons. The Journal of Molecular Diagnostics 2013 15, 401-412DOI: (10.1016/j.jmoldx.2013.02.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 A: SMART gel plot after on-chip amplification for 180 minutes, showing the detection of K103N sDNA at different concentrations (lanes 1, 2, 6, and 7), using a K103N probe in the absence (B−) or presence (B+) of beacons. No DNA (lanes 3 and 8) and low and high concentrations of WT single-stranded DNA (lanes 4, 5, 9, and 10) were also included as negative controls for detection with a K103N probe. B: SMART electropherogram of the gel plot shown in A, showing that the addition of molecular beacons has a significant inhibitory effect on probe amplification, especially for low starting concentrations of K103N sDNA. The Journal of Molecular Diagnostics 2013 15, 401-412DOI: (10.1016/j.jmoldx.2013.02.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 SMART assay results using off-chip amplification with real-time detection. A: WT sDNA and no DNA negative controls were included and plotted as background fluorescence (gray area). WT-specific SMART probes, WT primers, and WT molecular beacon were not included in this assay. The dashed black line indicates the threshold of detection or Tp for positive samples. SD was within 5% of results (error bars not shown). B: The Tp values are plotted versus the logarithmic scale of starting K103N sDNA concentration. SD was within 5% of results (error bars not shown). C: Histogram of normalized fluorescence of all sDNA and controls after 180 minutes’ reaction time. SD was within 5% of results (error bars not shown). The Journal of Molecular Diagnostics 2013 15, 401-412DOI: (10.1016/j.jmoldx.2013.02.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Off-chip real-time fluorescence of amplified K103N probe as a function of time. Total concentration of SMART probes (K103N + WT) were 1 pmol/L (6 × 108 copies/mL). SD was within 5% of results (error bars not shown). Background fluorescence is shown as a shaded gray area. The Journal of Molecular Diagnostics 2013 15, 401-412DOI: (10.1016/j.jmoldx.2013.02.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Off-chip real-time fluorescence of amplified WT probe as a function of time. Total concentration of SMART probes (K103N + WT) was 1 pmol/L (6 × 108 copies/mL). SD was within 5% of results (error bars not shown). Background fluorescence is shown as a shaded gray area. The Journal of Molecular Diagnostics 2013 15, 401-412DOI: (10.1016/j.jmoldx.2013.02.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Off-chip modeling of fluorescence versus K103N SMART probe concentration at high concentrations (3 × 107 to 1.5 × 108 copies/mL K103N SMART probe) (A) and low concentrations (≤3 × 107 copies/mL K103N SMART probe) (B). The Journal of Molecular Diagnostics 2013 15, 401-412DOI: (10.1016/j.jmoldx.2013.02.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 7 On-chip modeling of fluorescence versus K103N SMART probe concentration at high concentrations (3 × 107 to 1.5 × 108 copies/mL K103N SMART probe) (A) and low concentrations (≤3 × 107 copies/mL K103N SMART probe) (B). The Journal of Molecular Diagnostics 2013 15, 401-412DOI: (10.1016/j.jmoldx.2013.02.005) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions