Table 1. Trypanosoma cruzi PCR assays used for screening, discrete typing unit (DTU), and phylogenetic analyses.
| PCR assay1 | Forward/Reverse Primers & (Reaction conditions) | bp2 | Cycling conditions (‘ = minutes;” = seconds) | Cloning3 |
|---|---|---|---|---|
| S: TcZ nuclear 195bp satellite [31] | TcZ1: 5'-CGA GCT CTT GCC CAC ACG GGT GCT-3' / TcZ2: 5'-CCT CCA AGC AGC GGA TAG TTC AGG-3' (20μl: 1.9mM MgCl2, 0.2mM each dNTPs, 0.5μM each primer, 0.25U Taq & 4μl buffer) | 188 | 94° x 5’ (94° x 20”, 57° x 10”, 72° x 30”)35; 72° x 7’ | 1 |
| S: kDNA minicircle [32] | 121: 5'-AAA TAA TGT ACG GGK GAG ATG CAT GA-3' / 122: 5'-GGT TCG ATT GGG GTT GGT GTA ATA TA-3' (25μl: 1.5mM MgCl2, 0.2mM each dNTPs, 0.4μM each primer, 0.625U Taq & 5μl buffer) | 330 | 95° x 10’ (94° x 30”, 58° x 30”, 72° x 1’)35; 72° x 10’ | 1 |
| DTU: LSU rDNA [33] | D71: 5'-AAG GTG CGT CGA CAG TGT GG-3' / D72: 5'-TTT TCA GAA TGG CCG AAC AGT-3' (25μl: 1.5mM MgCl2, 0.1mM each dNTPs, 0.4μM each primer, 1U Taq & 5μl buffer) | varies | 94° x 3’ (94° x 1’, 60° x 1’, 72° x 1’)35; 72° x 10’ | 1 |
| DTU: HSP60 [34] | For: 5'-GTG GTA TGG G TGA CAT GTA C-3' / Rev: 5'-CGA GCA GCA G AGC GAA ACA T-3' (50μl: 2mM MgCl2, 0.1mM each dNTPs, 0.4μM each primer, 2U Taq & 13.3μl buffer) | 432–462 prior to RFLP4 | 94° x 3’ (94° x 30”, 52° x 30”, 72° x 1’)35; 72° x 10’ | 1 |
| DTU: Mini-exon [33] 5 | TC: 5’-CCC CCC TCC CAG GCC ACA CTG-3’ / TC1: 5’- GTG TCC GCC ACC TCC TTC GGG CC-3’ / TC2: 5’-CCT GCA GGC ACA CGT GTG TGT G-3’ (25μl: 1.5 MgCl2, 0.2mM each dNTPs, 1μM each primer, 1U Taq & 5μl buffer) | varies | 94° x 3’ (94° x 30”, 60° x 30”, 72° x 30”)27; 72° x 10’ | 1 |
| DTU: GPI [34] | For: 5'-GGC ATG TGA AGC TTT GAG GCC TTT TTC AG-3' / Rev: 5'-TGT AAG GGC CCA GTG AGA GCG TTC GTT GAA TAG C-3' (50μl: 2mM MgCl2, 0.1mM each dNTPs, 0.4μM each primer, 2U Taq & 13.3μl buffer) | 1264 prior to RFLP4 | 94° x 3’ (94° x 30”, 52° x 30”, 72° x 1’)35; 72° x 10’ | 1 |
| P: COII-ND1 maxicircle [35] | ND3.1A: 5’-GCT ACT ART TCA CTT TCA CAT TC-3’ / COII.2A: 5’-GCA TAA ATC CAT GTA AGA CMC CAC A-3’ (25μl: 1.5 MgCl2, 0.2mM each dNTPs, 1μM each primer, 1U Taq & 5μl buffer) | 1272 | 94° x 5’ (94° x 30”, 50° x 30”, 72° x 30”)30; 72° x 7’ | 2 |
| P: TR nuclear [35] | TR Y2S: 5’-ACT GGA GGC TGC TTG GAA CGC-3’ / TR Y2A: 5’-GGA TGC ACA CCR ATR GTG TTG T-3’ (25μl: 1.5 MgCl2, 0.2mM each dNTPs, 0.5μM each primer, 1U Taq & 5μl buffer) | 1335 | 94° x 5’ (94° x 1’, 55° x 1’, 72° x 1’)30; 72° x 5’ | 1 |
| P: RB19 nuclear [36] | RB19F: 5’-GCC TAC ACC GAG GAG TAC CA-3’ / RB19R: 5’-TTC TCC AAT CCC CAG ACT TG-3’ (25μl: 1.5 MgCl2, 0.2mM each dNTPs, 0.5μM each primer, 1U Taq & 5μl buffer) | 408 | 94° x 5’ (94° x 1’, 55° x 1’, 72° x 1’)30; 72° x 5’ | 3 |
1Bracketed numbers correspond to the original references for each assay. S = Screening; P = Phylogenetic
2The target base pair amplicon sizes; for some assays the size varies by the discrete typing unit as depicted in Fig 1
3Cloning methods were M13 PCR amplification followed by ExoSapIT (1), Qiagen MiniPrep method followed by EcoRI digestion (2), or direct sequencing without cloning (3).
4See Fig 1 for details on the restriction fragment length polymorphism (RFLP) banding pattern for HSP60 and GPI.
5Multiplex PCR assay with three primers.