LAMP primer sequences, sequence alignment comparisons, and LAMP primer sequence overlaps. (A) The Trypanosoma brucei gambiense (Tbg) and Trypanosoma brucei brucei (Tbb) isolates with each ribosomal RNA-internal transcribed spacer 2 (5.8S-ITS2) sequence used for their alignment include Tbg DAL 972 (GenBank accession no. AF306774), Tbg Suzena (AF406775), Tbg NW2 (AF306776), Tbg TH2 (AF306777), Tbb KP2 (AF306773), Tbb B8/18 (AF30677), Tbb STIB215 (AF306771), and Tbb H3 (AF306770). Using the online CLUSTAL-W program (www.genome.jp/tools/clustalw/), the first C3A fragment (461–464) on the 5.8S-ITS2 gene is conserved among T. brucei species isolated from humans and animals. The second fragment of C3A (557–560) is only conserved among Trypanosoma brucei gambiense subspecies. (B) The basic TBG4 LAMP primers consisting F3, B3, FIP, and BIP primers and the LF and LB loop primers are shown. (C) The 186-bp target area (gray shading) on the 5.8S-ITS2 gene where TBG4 (shown in blue) and TBG1 primers overlap. The C3A sequences (shown in red) and the dashed and solid lines show the TBG4 LAMP and TBG1 LAMP primer sets, respectively. The primers from each set greatly overlap except for TBG4-F3. B3 = outer backward primer; BIP = backward inner primer; FIP = forward inner primer; F3 = outer forward primer; LAMP = loop-mediated isothermal amplification; LB = loop backward; LF = loop forward.