TABLE 2.
PCR primers and conditions used for analysis of the sfp cluster and its flanking regions in EHEC O165:H25/NM
| PCR no. | Primera | Sequence (5′-3′) | Target | PCR conditionsb
|
PCR product size (bp) | ||
|---|---|---|---|---|---|---|---|
| Denaturing | Annealing | Extension | |||||
| I | wprom-3 | GCT GGA CGC CGG TGC TTA TT | Tn2501-sfpA | 94°C, 30 s | 61°C, 60 s | 72°C, 150 s | 1,900 |
| wprom-4 | CCA GAT TGC CGC TCG CTT CAG | ||||||
| II | IS2-1 | GTG ATA ACA AGC GTC TGG AAA | IS2-sfpA | 94°C, 30 s | 57°C, 60 s | 72°C, 60 s | 867 |
| wprom-4 | CCA GAT TGC CGC TCG CTT CAG | ||||||
| III | IS2-3 | CGA TGA CTG GAA GGA TAG | IS2-sfpA | 94°C, 30 s | 50°C, 60 s | 72°C, 90 s | 1,161 |
| sfpA-2 | AGC CAG TTC CTT GAC ATC | ||||||
| IV | sfpA-U | AGC CAA GGC CAA GGG ATT ATT A | sfpA | 94°C, 30 s | 59°C, 60 s | 72°C, 60 s | 440 |
| sfpA-L | TTA GCA ACA GCA GTG AAG TCT C | ||||||
| V | sfpH-1 | CCA TCG CCC TCC TTT CCT CT | sfpH | 94°C, 30 s | 58°C, 60 s | 72°C, 60 s | 425 |
| sfpH-2 | TCC AGC CCC TCT TCA TTT CC | ||||||
| VI | sfpC-5 | TAT GCG TTG TTG TTT TCC | sfpC 5′ end | 94°C, 30 s | 55°C, 60 s | 72°C, 150 s | 1,643 |
| sfpC-6 | TCC CGT TTC TGG TAT TTC | ||||||
| VII | sfpC-1 | CGG GCA TCG GAC TAT TAC AC | sfpC 3′ end | 94°C, 30s | 58°C, 60 s | 72°C, 60 s | 787 |
| sfpC-2 | CCA CCA TCC CCA GTT CAC G | ||||||
| VIII | sfpD-1 | CGC CAA TGA TAA CAA ACA G | sfpD | 94°C, 30 s | 52°C, 60 s | 72°C, 60 s | 410 |
| sfpD-2 | TAC CAC CAA TGC CAA TCA C | ||||||
| IX | sfpJ-1 | ATG AGC AGG ACA ACA ACA GG | sfpJ | 94°C, 30 s | 52°C, 60 s | 72°C, 60 s | 251 |
| sfpJ-2 | TAT TTT CCG TTA TCC TCC AG | ||||||
| X | sfpG-1 | CGT AGT GAC TTG GAT GCT G | sfpG | 94°C, 30s | 52°C, 60s | 72°C, 60s | 267 |
| sfpG-2 | TTG CCA TCC ATT ACC ATT C | ||||||
| XI | sfpA-U | AGC CAA GGC CAA GGG ATT ATT A | sfpA-sfpH | 94°C, 30s | 59°C, 60s | 72°C, 60s | 1,026 |
| sfpH-2 | TCC AGC CCC TCT TCA TTT CC | ||||||
| XII | sfpH-1 | CCA TCG CCC TCC TTT CCT CT | sfpH-sfpC | 94°C, 30s | 59°C, 60s | 72°C, 60s | 733 |
| sfpC-3 | CGC CAG CCA GGG AAA ACA ACA A | ||||||
| XIII | sfpC-4 | GTG GGG AGT TGG TGT AGT GA | sfpC-sfpD | 94°C, 30s | 56°C, 60s | 72°C, 60s | 1,030 |
| sfpD-2 | TAC CAC CAA TGC CAA TCA C | ||||||
| XIV | sfpD-1 | CGC CAA TGA TAA CAA ACA G | sfpD-sfpJ | 94°C, 30s | 52°C, 60s | 72°C, 60s | 875 |
| sfpJ-2 | TAT TTT CCG TTA TCC TCC AG | ||||||
| XV | sfpJ-1 | ATG AGC AGG ACA ACA ACA GG | sfpJ-sfpG | 94°C, 30s | 52°C, 60s | 72°C, 150s | 1,748 |
| sfpG-2 | TTG CCA TCC ATT ACC ATT C | ||||||
| XVI | sfpG-3 | GCA TAA CAA TAA ACT CCA AA | sfpG | 94°C, 30 s | 48°C, 60 s | 72°C, 60 s | 588 |
| Gfla-1 | TGA CAA GCA AAC TGA TTA CC | Flank 1 | |||||
| XVII | sfpG-3 | GCA TAA CAA TAA ACT CCA AA | sfpG | 94°C, 30 s | 50°C, 60 s | 72°C, 120 s | 1,325 |
| Gfla-3 | CAT TGC TCC CTT CAT AAA A | Flank 2 | |||||
| XVIII | sfpG-5 | GGA TCG TAA TGC TGT TTC A | sfpG-repA | 94°C, 30 s | 51°C, 60 s | 72°C, 150 s | 1,343 |
| Gfla-4 | AAG CAC CGA ATG ATG AGA G | ||||||
| XIX | sfpG-5 | GGA TCG TAA TGC TGT TTC A | sfpG-repA | 94°C, 30 s | 51°C, 60 s | 72°C, 180 s | 1,768 |
| Gfla-5 | TAC CGT GAT CTC CTT TTC C | ||||||
a
The primers were derived from the respective genes of the sfp cluster and from the sfp flanking regions in SF EHEC O157:NM strain 3072/96 (GenBank accession no. AF401292). Except for primer pairs wprom-3/wprom-4 and sfpA-U/sfpA-L, which were described previously (11), all primers were designed for this study.
b
All PCRs included 30 cycles as given in the table, preceded by an initial denaturation (94°C, 5 min) and followed by a final extension (72°C, 5 min).