Table 3 PCR conditions used to differentiate T. equiperdum and T. evansi
Target | Primers | Primer sequences | Amplicon length (bp) | Reaction mixturea | Cycling conditions | Adapted from |
|---|---|---|---|---|---|---|
VSG RoTat 1.2 | ILO7957 ILO8091 | 5′-GCC ACC ACG GCG AAA GAC-3′ 5′-TAA TCA GTG TGG TGT GC-3′ | 488 | a | 95 °C for 5 min and 35 cycles of 30 s at 94 °C, 30 s at 58 °C, 30 s at 72 °C and final extension for 5 min at 72 °C | [55] |
VSG RoTat 1.2 | RoTat1.2-F RoTat1.2-R | 5′-GCGGGGTGTTTAAAGCAATA-3′ 5′-ATTAGTGCTGCGTGTGTTCG-3’ | 205 | a | 95 °C for 15 min and 40 cycles of 30 s at 94 °C, 30 s at 59 °C, 30 s at 72 °C and final extension for 5 min at 72 °C. | [56] |
Maxicircle A6 | Forward Reverse | 5′AAAAATAAGTATTTTGATATTATTAAAG-3′ 5′-TATTATTAACTTATTTGATC-3′ | 381 | b | 95 °C for 5 min and 30 cycles of 94 °C for 1 min, 54 °C for 1 min and 72 °C for 30s followed by a final elongation step at 72 °C for 8 min | [57] |
Maxicircle ND4 | Forward Reverse | 5′-TGTGTGACTACCAGAGAT-3′ 5′ -ATCCTATACCCGTGTGTA-3 | 256 | b | Idem as above | [57] |
Maxicircle ND5 | Forward Reverse | 5′-TGGGTTTATATCAGGTTCATTTATG-3′ 5′ -CCCTAATAATCTCATCCGCAGTACG-3′ | 400 | b | Idem as above | [58] |
Maxicircle ND7 | Forward Reverse | 5′-ATGACTACATGATAAGTA-3′ 5′ -CGGAAGACATTGTTCTACAC-3′ | 167 | b | Idem as above | [57] |