Table 3.
Nested PCR primers and cycling conditions used to amplify two smaller, overlapping fragments of large fragments no. 2, 3, and 4
| PCR | Forward (F) and reverse (R) PCR primers | Modified after | Fragment size (bp) | Cycle conditions: temperature (°C)/time (s) for denaturation, annealing and extension steps |
|---|---|---|---|---|
| 2.1 | F: ATAAAGAATATTATTTATAAGAACGG | 1.315 | 94/30, 48/30, 72/80 | |
| R: GGATGTCCAAAGAACCAGAA | ||||
| 2.2 | F: ACTGGATGGACTTTATATCC | 940 | 94/30, 48/30, 72/60 | |
| R: CCAAGGAAATGCATAGGTAA | ||||
| 3.1 | F: GCTTTACATGATACATATTATGTAATTGC | = 3180F (Perkins 2008) | 1.080 | 94/30, 53/30, 72/60 |
| R: GCATTATCTGGATGTGATAATGGT | = HaemR2 (Bensch et al. 2000) | |||
| 3.2 | F: TTACCTTGGGGTCAAATGAG | 1.190 | 94/30, 50/30, 72/80 | |
| R: GAATATAGACGGTTTTCTGCG | ||||
| 4.1 | F: GGCAAGTTAAAGAAGTTCTGGTTT | 1.070 | 94/30, 53/30, 72/60 | |
| R: TTGAATGGAGCACTGGATTGG | 5939R (Perkins 2008) | |||
| 4.2 | F: ATCCTTAAATCTCGTAACCATGC | F2 (Pacheco et al. 2018b) | 1.050 | 94/30, 56/30, 72/60 |
| R: GGGAAGTGTGTTTCCATAGAAACCTTC | 626R (Perkins 2008) |
All outer reactions included an initial denaturation period of 4 min at 94 °C and 25 cycles. Nested reactions included an initial denaturation period of 4 min at 94 °C and 35 cycles. All reactions included a final extension period of 7 min at 72 °C