Table 1.
Universal and specific primers designed to detect microsporidian species
| Primer | Sequence (5′–3′) | Annealing Temperature | Specificity |
|---|---|---|---|
| V1 | CAC CAG GTT GAT TCT GCC TGA C | 62 °C | universal (Zhu et al. 1993, McClymont et al. 2005) |
| 1342R | ACG GGC GGT GTG TAC AAA GAA CAG | ||
| Dict11 F | CGA CGT AAA CCT TTT GGT GCA R | 60 °C | Dictyocoela duebenum/ D. muelleri (Gr/GpC) |
| Dict11 R | TYT CTT CCG CAA TAC TAA AAA ATT AAT AC | ||
| Dict14 F | GGG CGA TTT ATT TGT TCT CCT GT | 58 °C | Dictyocoela duebenum/ D. muelleri (Gf/GpE) |
| Dict14 R | GAT TTC TCT TCC GCA ATA CCA AAT YG | ||
| Dict16/17 F | ATT GAT TAA RGA ACG AGC AGG GTT AG | 62 °C | Dictyocoela duebenum/ D. muelleri (GpC/Dh) |
| Dict16/17 R | TCT TCC GCA AYA CMG CCA CA | ||
| Dict15 F | TTT TAA TCG TGG CGT AAA CCA TK | 62 °C | D. berillonum |
| Dict15 R | CTC TTC CGC AAT ACA GAA TAC CAT AC | ||
| Mic505 F | CAT CAA CTA ACT TTG GGA AAC TAA G | 62 °C | Microsporidium sp. 505 |
| Mic505 R | TGG CCT CCC ACA CAT TCC GAG TG | ||
| Mic 515 F | GGC GAT CTA ACC TCG GCA TCG GAT AAC C | 62 °C | Microsporidium sp. 515 |
| Mic 515 R | TGG CTT CCC ACC CAT TCC GAG C | ||
| Mic 3 F | CAG TAA TGT TGC GAT GAT TTG GTC | 58 °C | Microsporidium sp. I |
| Mic 3 R | CAG TAA ATA CTC CAC AGT ATC TTA C | ||
| Mic4 F | TAC GGC TAA GAC GTG GAC | 58 °C | Microsporidium sp. nov. RR1 |
| Mic4 R | CAA TCC TAT TGC CAT CAT CTG | ||
| Mic6 F | AGG ACC GAC GGC AAA GAA GTC | 60 °C | Microsporidium sp. nov. RR2 |
| Mic6 R | CAG GAG ATC TCA CCC ATT CAG | ||
| Mic7 F | ACA GTT ATA ATT TAC TCG TAG ATC | 58 °C | Microsporidium sp. BPAR3 |
| Mic7 R | TAC TCG CAA GCA TGT GCT CA | ||
| Mic18/19 F | ATA GAG GCG GTA GTA ATG AGA CGT A | 58 °C | C. dikerogammari/ Microsporidium sp. G |
| Mic18/19 R | TTT AAC CAT AAA ATC ACT TCA CTC |
PCR program for all primers was: 98 °C for 3 min, 35 cycles of 98 °C-10 s, [annealing temp. see table]-10 s, 72 °C-15 s (10 s for Dict16/17, 20 s for V1/1342R) and final elongation at 72 °C for 3 min