Figure 2.

Selectivity and sensitivity of the dual luciferase assay in B. burgdorferi. (A) B. burgdorferi clones were grown to mid-logarithmic phase, and the in vitro luciferase assay performed with 700 μM D-luciferin or 3.5 mM coelenterazine. Relative luciferase units were normalized to optical density at 600 nm (OD₆₀₀) and presented as the mean relative luciferase units/OD₆₀₀ ± standard deviation for biological triplicate samples. The data were square root transformed and analyzed with a one-way ANOVA followed by Dunnett's multiple comparison test compared to B. burgdorferi containing the promoterless fluc_Bb (+pJSB161) for each substrate. Unless indicated, means were not significantly different from the control. Significant differences are indicated with asterisks (^****p ≤ 0.0001). (B) Mid-logarithmic phase grown B. burgdorferi expressing both flaBp-rluc_Bb and flaBp-fluc_Bb (+pCFA801) were serial diluted from 2 × 10⁸ to 2 × 10⁰ spirochetes, and incubated with 3.5 mM coelenterazine. The limit of detection (LoD) was established as the mean relative luciferase units for PBS alone plus 3 standard deviations (gray dotted line). The limit of quantitation (LoQ) was established as the mean relative luciferase units for PBS alone plus 10 standard deviations (red dotted line). Data are presented as the mean relative luciferase units ± standard deviation for biological triplicate samples.