Figure 4. Testing of spiked samples with the semi-nested duplex real-time PCR assay.

The analytical sensitivity of the assay was estimated through the use of solutions of DNA extracted from MTC-free tissue samples spiked with serially diluted cellular suspensions of M. tuberculosis ATCC 25177 (ranging from 10⁷ to 10° cells/ml of tissue homogenate). The figure illustrates the IS6110 (solid lines) and mammal β-actin gene (dashed lines) targeted amplification curves obtained in FAM and Cy5.5 channels, respectively, for the several dilutions: 10⁷ (yellow line), 10⁵ (black line), 10³ (brown line), 10¹ (green line) and 10° (blue line) cells. Positive control: M. tuberculosis ATCC 25177 culture (red line); Negative controls: unspiked tissue homogenate (orange line) and non-template control (grey line). RFU - Relative Fluorescence Units.