Table 1 Primer pairs, targets and conditions commonly used for MAP detection
From: MAP, Johne’s disease and the microbiome; current knowledge and future considerations
Primer pair | Target | Size of product (bp) | PCR conditions | References |
|---|---|---|---|---|
J5A (Forward 5′-ATGTGGTTGCTGTGTTGGATGG-3′) J5B (Reverse 5′-CCGCCGCAATCAACTCCAG-3′) | IS900 | 298 | 94 °C for 5 min; [94 °C for 30 s, 66 °C for 30 s, and 72 °C for 1 min] × 4 cycles; [94 °C for 30 s, 64 °C for 30 s, and 72 °C for 1 min] × 4 cycles; [94 °C for 30 s, 62 °C for 30 s, and 72 °C for 1 min] × 4 cycles, [94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min] × 24 cycles, and a final extension at 72 °C for 7 min | [98] |
IS900qPCRF(Forward 5′-GATGGCCGAAGGAGATTG-3′) IS900qPCRR(Reverse 5′- CACAACCACCTCCGTAACC-3′) | IS900 | 145 | 37 °C for 10 min, followed by initial denaturation at 95 °C for 15 min and 47 cycles of 95 °C for 5 s and 60 °C for 40 s | [99] |
F57qPCRF (Forward 5′-GCCCATTTCATCGATACCC-3′) F57qPCRR (Reverse 5′-GTACCGAATGTTGTTGTCAC-3′) | F57 | 147 | 37 °C for 10 min, followed by initial denaturation at 95 °C for 15 min and 47 cycles of 95 °C for 5 s and 60 °C for 40 s | [99] |
(Forward 5′ -CCGCTAATTGAGAGATGCGATTGG-3') (Reverse 5′ -AATCAACTCCAGCAGCGCGGCCTCG-3′) | IS900 | 229 | 1 cycle at 94 °C for 5 min and 20 cycles at 94 °C for 45 s, 58 °C for 1 min, and 72 °C for 2 min, followed by a final extension cycle at 72 °C for 7 min | [100] |
f57_F (Forward 5′-TTG GAC GAT CCG AAT ATG T-3′) f57_R (Reverse 5′-AGT GGG AGG CGT ACC A-3′) | F57 | 254 | 1 cycle of pre-incubation: 95 °C for 10 min. Followed by 40 cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C for 20 s | |
mbtA_F3 (Forward 5′–CTC CCG CAA CTC GGT CAC–3) MAP2179_R3 (Reverse 5′–CAC AGC CAG GTG TGA AAG–3′) | mbtA | 307 | 1 cycle of pre-incubation: 95 °C for 10 min. Followed by 40 cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C for 20 s | [97] |
P90 (Forward 5′-GAA GGG TGT TCG GGG CCG TCG GCC TTA GG- 3′) P91 (Reverse 5′-GGC GTT GAG GTC GAT CGC CCA CGT GAC-3') | IS900 | 394 | 4 min of initial denaturation at 95 °C, 37 cycles of 95 °C for 30 s, 59.5 °C for 30 s and 72 °C for 30 s, followed by a final elongation at 72 °C for 4 min | |
AV1 (Forward 5'-ATGTGGTTGCTGTGTTGGATGG-3') AV2 (Reverse 5'-CCGCCGCAATCAACTCCAG-3') | IS900 | N/A | 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 58 °C for 1 min and 72 °C for 1 min |