, 1999; Eddyani et al., 2004; Kotlowski et al., 2004; Johnson et al., 2007), indicating

that M. ulcerans is probably present in such samples. However, other closely related Mycobacterium species including Mycobacterium liflandii, Mycobacterium Pseudoshottsii, and mycolactone-producing Mycobacterium marinum strains (Stinear et al., 1999; Stragier et al., 2007) have been found to harbor IS2404. Thus, the conventional IS2404 PCR assay cannot be relied upon for the specific detection of M. ulcerans. In order to increase specificity, facilitate rapid analysis of specimens, and to interpret the results of both environmental and clinical specimens Ivacaftor purchase with certainty, Fyfe et al. (2007) developed two TaqMan Multiplex real-time PCR assays targeting three independent repeated sequences in the M. ulcerans genome, two multicopy insertion sequences (IS2404, IS2606), and a multicopy sequence encoding the ketoreductase

B domain (KR-B). These real-time PCR assays quantify the copy number of the targets, allowing Y-27632 supplier the differentiation of M. ulcerans from other IS2404-containing mycobacteria. Moreover, the assay allows for the control of PCR inhibitors such as humic and fulvic acids, commonly present in environmental samples. In spite of its advantages for the analysis of clinical and environmental samples (high throughput, high sensitivity and specificity, less prone to contamination, and Fluorouracil chemical structure inhibition control), facilities for real-time PCR are available only in a few research laboratories in West-African BU-endemic countries, including Ghana. However, swift analysis of environmental samples could be crucial

in the search for the M. ulcerans reservoir. Therefore, the current study describes the first application of real-time PCR for the detection of M. ulcerans in environmental samples at the Noguchi Memorial Institute for Medical Research (NMIMR) in Accra, Ghana. Both the acquisition of these technologies through international technology transfer and their diffusion will foster effective technological change as follow-on innovation and adaptation occurs. The real-time PCR assays were carried out as described by Fyfe et al. (2007). Briefly, IS2404/internal positive control (IPC) mixtures contained 1 μL of template DNA, 0.9 μM of each primer, 0.25 μM of the probe, 1 × TaqMan® Universal PCR Master Mix (Applied Biosystems, Foster City, CA), and TaqMan exogenous IPC reagents (Applied Biosystems) in a total volume of 25 μL. IS2606/KR assays were preformed on IS2404-positive samples in a similar multiplex way without IPC. Detection was performed on a 7300 real-time PCR System (Applied Biosystems) using the following thermal profile: one cycle of 50 °C for 2 min, one cycle of 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min.