Rapid Detection of TEM-Type Extended-Spectrum β-Lactamase (ESBL) Mutations Using Lights-On/Lights-Off Probes with Single-Stranded DNA Amplification  Kenneth E. Pierce, Harald Peter, Till T. Bachmann, Carmelo Volpe, Rohit Mistry, John E. Rice, Lawrence J. Wangh  The Journal of Molecular Diagnostics  Volume 15, Issue 3, Pages 291-298 (May 2013) DOI: 10.1016/j.jmoldx.2013.02.002 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic representation of the interactions between lights-on probes (short lines with both open and solid circles), lights-off probes (short lines with only one solid circle), and the single-stranded amplification product (continuous long line). At low temperatures, all eight probes are hybridized to the nonmutated TEM-1 target and the quencher (solid circle) on the lights-off probes prevents emission from the fluorescent molecule (open circle) of the adjacent lights-on probe. As temperature increases, each lights-off probe melts from the product before the adjacent lights-on probe does, increasing fluorescence until the latter also melts and fluorescence is reduced by interaction with its own quencher. Probes are designed with melting temperatures that give sequential melts of the probes from site 69, site 104, site 164, and finally site 238–240. ESBL mutations at a given site reduce the Tm of the lights-off probe at that site, which results in much higher fluorescence from the paired lights-on probe at a lower temperature and changes the overall melt contour. EP, excess primer; LP, limiting primer; X, site of mutation. The Journal of Molecular Diagnostics 2013 15, 291-298DOI: (10.1016/j.jmoldx.2013.02.002) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Fluorescence signal melt contours reflect the sequence variations of different TEM genes and the number of starting copies. Fluorescence signal melt contours after amplification of 100,000 targets (black), 10,000 targets (dark gray), or 1000 targets (light gray) of synthetic genes TEM-1 (A), TEM-3 (B), and TEM-152 (C). A: For TEM-1, each fluorescence maximum (arrows) is reached as a lights-off probe melts from the single-stranded amplification product. Subsequent melting of the paired lights-on probe results in decreasing fluorescence. Amino-acid positions of ESBL mutations tested by the probes at the corresponding temperature are indicated above the maxima. B and C: Fluorescent signals for TEM-3 and TEM-152 are relatively high, and maxima are shifted to the left, compared with TEM-1, because of lower-temperature melting of the lights-off probes over mutation sites. Fluorescence data are expressed in relative fluorescence units. Four replicate samples were tested at each concentration. The Journal of Molecular Diagnostics 2013 15, 291-298DOI: (10.1016/j.jmoldx.2013.02.002) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Fluorescence signal melt contours are unaffected by the presence of human genomic DNA. Fluorescence signal melt contours after amplification of 1000 targets of synthetic genes TEM-1 (A), TEM-3 (B), and TEM-152 (C) in the presence of 6 ng (dark gray) or 60 ng human genomic DNA (light gray), or without human genomic DNA (black). Four replicate samples were tested for each genomic DNA concentration with each TEM variant. The Journal of Molecular Diagnostics 2013 15, 291-298DOI: (10.1016/j.jmoldx.2013.02.002) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Fluorescence signal melt contours after amplification of different TEM genes from reference bacterial samples. A: TEM-3 and TEM-52 have ESBL mutations at sites 104 and 238 and show similar contours, with strong signal fluorescence. B: TEM-12 shows increased fluorescence around 55°C, relative to TEM-1, consistent with an ESBL mutation at site 164. TEM-132 has increased fluorescence around 65°C, consistent with the ESBL mutations at site 240, and also increased fluorescence around 52°C, because of mutations hybridized by the site 164 lights-on probe and lights-off probe. C: TEM-40, with an IRT mutation at site 69, shows greatly increased fluorescence for that site. TEM-34, with an IRT mutation at site 69, shows a 1°C shift in the fluorescence maximum relative to TEM-1 and a moderate increase in the maximum (39°C) to minimum (44°C) change in fluorescence. TEM-1 contours (dashed lines) are shown for comparison. All samples were tested in duplicate. The Journal of Molecular Diagnostics 2013 15, 291-298DOI: (10.1016/j.jmoldx.2013.02.002) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Fluorescent derivative plots for the samples shown in Figure 4C. Differences in the patterns from TEM-1 (dashed lines), TEM-34, and TEM-40 synthetic gene samples can be resolved using these fluorescent signatures. The values at 40°C (approximately the TEM-1 zero crossing point) are significantly different for each TEM gene variant (P = 0.0001, t-test). The Journal of Molecular Diagnostics 2013 15, 291-298DOI: (10.1016/j.jmoldx.2013.02.002) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions