Table 2 PCR primers and cycling protocols to amplify target sequences from Cryptosporidium, Cyclospora, Giardia and Entamoeba
Parasite | PCR target | Size (bp) | Primer | Reference | Cycling protocol | Reference |
|---|---|---|---|---|---|---|
Cyclospora | SSU | 1000 | ExCycF (forward: 5′-AATGTAAAACCCTTCCAGAGTAAC-3′) | [90] | 94 °C/ 5 min, followed by 35 cycles of 94 °C/ 45 s, 55 °C/ 45 s and 72 °C/ 1 min, with a final extension of 72 °C/ 10 min | [91] |
|  |  |  | ExCycR (reverse: 5′-GCAATAATCTATCCCCATCACG-3′) | |||
|  |  | 500 | NesCycF (forward: 5′-AATTCCAGCTCCAATAGTGTAT-3′) | Secondary amplification was achieved employing identical PCR conditions to those used in the primary PCR | ||
|  |  |  | NesCycR (reverse: 5′-CAGGAGAAGCCAAGGTAGGCRTTT-3′) | |||
Cryptosporidium | SSU | 824–864 | XF2 (forward: 5′-GGAAGGGTTGTATTTATTAGATAAAG-3′) | [92] | 94 °C/ 5 min, followed by 35 cycles of 94 °C/ 45 s, 60 °C/ 45 s and 72 °C/ 1 min, with a final extension of 72 °C/ 10 min | [92] |
|  |  |  | XR2 (reverse: 5′-AAGGAGTAAGGAACAACCTCCA-3′) | |||
|  |  | 298 | 18SiF (forward: 5′-AGTGACAAGAAATAACAATACAGG-3′) | [93] | 94 °C/ 5 min, followed by 35 cycles of 94 °C/ 45 s, 50 °C/ 45 s and 72 °C/ 1 min, with a final extension of 72 °C/ 10 min | [93] |
|  |  |  | 18SiR (reverse: 5′-CCTGCTTTAAGCACTCTAATTTTC-3′) | |||
|  | gp60 | 1000 | AL3531 (forward: 5′-ATAGTCTCCGCTGTATTC-3′) | [94] | 94 °C/ 5 min, followed by 35 cycles of 94 °C/ 45 s, 50 °C/ 45 s and 72 °C/ 1 min, with a final extension of 72 °C/ 10 min | [95] |
|  |  |  | AL3535 (reverse: 5′-GGAAGGAACGATGTATCT-3′) | [96] | ||
|  |  | 457 | AL3532 (forward: 5′-TCCGCTGTATTCTCAGCC-3′) | [94] | Secondary amplification was achieved employing identical PCR conditions to those used in the primary PCR | |
|  |  |  | AL3533 (reverse: 5′-GAGATATATCTTGGTGCG-3′) | |||
Giardia | tpi | 605 | AL3543 (forward: 5′-AAATTATGCCTGCTCGTCG-3′) | [84] | 94 °C/ 5 min, followed by 35 cycles of 94 °C/ 45 s, 50 °C/ 45 s and 72 °C/ 1 min, with a final extension of 72 °C/ 10 min | [84] |
|  |  |  | AL3546 (reverse: 5′-CAAACCTTTTCCGCAAACC-3′) | |||
|  |  | 530 | AL3544 (forward: 5′-CCCTTCATCGGTGGTAACTT-3′) | Secondary amplification was achieved employing identical PCR conditions to those used in the primary PCR | ||
|  |  |  | AL3545 (reverse: 5′-GTGGCCACCACTCCCGTGCC-3′) | |||
|  | bg | 753 | G7 (forward: 5′-AAGCCCGACGACCTCACCCGCAGTGC-3′) | [63] | 94 °C/ 5 min, followed by 35 cycles of 94 °C/ 30 s, 65 °C/ 30 s and 72 °C/ 1 min, with a final extension of 72 °C/ 7 min | [63] |
|  |  |  | G759 (reverse: 5′-GAGGCCGCCCTGGATCTTCGAGACGAC-3′) | |||
|  |  | 511 | bgiF (forward: 5′-GAACGAACGAGATCGAGGTCCG-3′) | [97] | 95 °C/ 15 min, followed by 35 cycles of 95 °C/ 30 s, 55 °C/ 30 s and 72 °C/ 1 min, with a final extension of 72 °C/ 7 min | [97] |
|  |  |  | bgiR (reverse: 5′-CTCGACGAGCTTCGTGTT-3′) | |||
|  | gdh | 786 | Ghd1 (forward: 5′-TTCCGTRTYCAGTACAACTC-3′) | [53] | 94 °C/ 2 min, followed by 35 cycles of 94 °C/ 30 s, 50 °C/ 30 s and 72 °C/ 1 min, with a final extension of 72 °C/ 7 min | [53] |
|  |  |  | Gdh2 (reverse: 5′-ACCTCGTTCTGRGTGGCGCA-3′) | |||
|  |  | 530 | Gdh3 (forward: 5′-ATGACYGAGCTYCAGAGGCACGT-3′) | Secondary amplification was achieved employing identical PCR conditions to those used in the primary PCR | ||
|  |  |  | Gdh4 (reverse: 5′-GTGGCGCARGGCATGATGCA-3′) | |||
|  | SSU | 497 | Gia2029 (forward: 5′-AAGTGTGGTGCAGACGGACTC-3′) | [98] | 94 °C/ 4 min, followed by 35 cycles of 96 °C/ 45 s, 55 °C/ 30 s and 72 °C/ 45 s, with a final extension of 72 °C/ 4 min | [98] |
|  |  |  | Gia2150c (reverse: 5′-CTGCTGCCGTCCTTGGATGT-3′) |  | ||
|  |  | 292 | RH11 (forward: 5′-CATCCGGTCGATCCTGCC-3′) | [99] | 94 °C/ 4 min, followed by 35 cycles of 96 °C/ 45 s, 59 °C/ 30 s and 72 °C/ 45 s, with a final extension of 72 °C/ 4 min | |
|  |  |  | RH4 (reverse: 5′-AGTCGAACCCTGATTCTCCGCCAGG-3′) |  | ||
Entamoeba | SSU |  | JVC (forward: 5′-GTTGATCCTGCCAGTATTATATG-3′) | [100] | 95 °C/ 5 min, followed by 40 cycles of 95 °C/ 30 s, 57 °C/ 30 s and 72 °C/ 1 min, with a final extension of 72 °C/ 4 min | [100] |
|  |  |  | DSPR2 (reverse: 5′-CACTATTGGAGCTGGAATTAC-3′) |