Skip to main content

Table 3 PCR conditions used to differentiate T. equiperdum and T. evansi

From: Histopathological lesions in reproductive organs, distal spinal cord and peripheral nerves of horses naturally infected with Trypanosoma equiperdum

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]
  1. aReaction mixture (a): 25 μl containing 25 ng DNA, 1x Green GoTaq G2 Flexi buffer, 2 mM of MgCl2, 0.2 mM of each dNTPs, 0.5 μM of each primer, 1.25 U GoTaqG2 Flexi DNA polymerase. Reaction mixture (b): 25 μl containing 25 ng DNA, 1x Green GoTaq G2 Flexi buffer, 2 mM of MgCl2, 0.2 mM of each dNTPs, 1 μM of each primer, 1.25 U GoTaqG2 Flexi DNA polymeras