TABLE 3.
PCR primers and conditions for amplification of virulence genes
| Target gene | Primer designation | Primer sequence (5′-3′) | PCR conditionsa | PCR product length (bp) | Reference |
|---|---|---|---|---|---|
| bfp (bundle-forming pilus) | EP1 | AAT GGT GCT TGC GCT TGC TGC | 94°C, 30 s; 61°C, 60 s; 72°C, 120 s | 397 | 26 |
| EP2 | GGC GCT TTA TCC AAC CTG GTA | ||||
| cdt (cytolethal distending toxin) | cdtS1/2b | GAA AG(A)T AAA TGG AAT(C) AT(C)A A(C)AT GTC CG | 94°C, 30 s; 52°C, 60 s; 72°C, 60 s | 466 | 59 |
| cdtAS1/2b | AAA TCA(T) CCA(T) A(G)G(C)A ATC ATC CAG TTA | ||||
| cnf (cytotoxic necrotizing factor) | cnf-sense | ATC TTA TAC TGG ATG GGA TCA TCT TGG | 94°C, 45 s; 55°C, 45 s; 72°C, 60 s | 974 | 59 |
| cnf-antisense | GCA GAA CGA CGT TCT TCA TAA GTA TC | ||||
| eae (all intimin genes) | SK1 | CCC GAA TTC GGC ACA AGC ATA AGC | 94°C, 30 s; 52°C, 60 s; 72°C, 45 s | 881 | 42, 64, 52 |
| SK2 | CCC GGA TCC GTC TCG CCA GTA TTC G | ||||
| Subtyping: eae β | SK1 and LP4 | CCC GTG ATA CCA GTA CCA ATT ACG GTC | 94°C, 45 s; 54°C, 60 s; 72°C, 150 s | 2,287 | |
| Subtyping: eae ɛ | SK1 and LP5 | AGC TCA CTC GTA GAT GAC GGC AAG CG | 94°C, 30 s; 45°C, 60 s; 72°C, 120 s | 2,608 | |
| E-hly (EHEC-hemolysin) | eha1 | GGT GCA GCA GAA AAA GTT GTA G | 94°C, 30 s; 57°C, 90 s; 72°C, 90 s | 1,551 | 48 |
| eha4 | TCT CGC CTG ATA GTA TTT GGT A | ||||
| espP (serine protease gene) | espA | AAA CAG CAG GCA CTT GAA CG | 94°C, 30 s; 56°C, 60 s; 72°C, 150 s | 1,830 | 16 |
| espB | GGA GTC GTC AGT CAG TAG AT | ||||
| etpD (part of type II secretion pathway) | D1 | CGT CAG GAG GAT GTT CAG | 94°C, 30 s; 52°C, 60 s; 72°C, 70 s | 1,062 | 50 |
| D13R | CGA CTG CAC CTG TTC CTG ATT A | ||||
| iutA (aerobactin receptor) | aero-f | GGC TGG ACA TCA TGG GAA CTG G | 94°C, 40 s; 67°C, 60 s; 72°C, 40 s | 280 | 30 |
| aero-r | CGT CGG GAA CGG GTA GAA TCG | ||||
| katP (catalase-peroxidase gene) | wkatB | CTT CCT GTT CTG CTG ATT CTT CTG G | 94°C, 30 s; 56°C, 60 s; 72°C, 150 s | 2,125 | 15 |
| wkatF | AAC TTA TTT CTC GCA TCA TCC | ||||
| paa (E. coli porcine attaching-effacing-associated protein) | 155-F1 | ATG AGG AAC ATA ATG GCA GG | 94°C, 30 s; 60°C, 30 s; 72°C, 30 s | 350 | 2, 6 |
| 155-R1 | TCT GGT CAG GTC GTC AAT AC | Accession no. U82533 | |||
| stx (Shiga toxin 1 and 2, all variants) | Lin-down | TTT GAT TGT TAC AGT CAT | 94°C, 60 s; 43°C, 90 s; 72°C, 90 s | ∼900 | 5, 33 |
| Lin-up | GAA CGA AAT AAT TTA TAT GT | ||||
| cif (cell cycle-inhibiting factor) | Cif-int-s | AAC AGA TGG CAA CAG ACT GG | 94°C, 30 s; 57°C, 60 s; 72°C, 30 s | 383 | 42 |
| Cif-int-as | AGT CAA TGC TTT ATG CGT CAT | ||||
| Cif-rbs-s | CGT GAA GGA GTG AGA TAT GAA AGA CAT TAC C | 94°C, 30 s; 53°C, 60 s; 72°C, 30 s | 948 | ||
| Cif-as | CTG AAT CAT TTT ACC GTA TGG | ||||
| φ-cif/chromosome | phage-s | TCG ATA CAA CAG AGA CAA ATG | 94°C, 30 s; 54°C, 30 s; 68°C, 240 sc | 4,993 | 42 |
| YbhB-sens | ATA ATA TTT CAC CGC ATC TGG | ||||
| efa1 5′ region (EHEC factor for adherence) | Efa1 fwd | TGG GCA GAA CAT TTT CAC CAG TTC | 94°C, 30 s; 58°C, 30 s; 72°C, 30 s | 725 | 40 |
| Efa1 rev | CTT TCA GGT GGG GAA CCA TAT GGC | ||||
| efa1 3′ region (EHEC factor for adherence) | Efa1 3′ fwd | TGC GCA CAA TTG ACT ACA GAG GAA | 94°C, 30 s; 58°C, 30 s; 72°C, 30 s | 692 | 40 |
| Efa1 3′ rev | ATA CGA CCA TCA GGG GAA TCA C |
a
30 cycles of amplification and a final extension step of 72°C for 5 min were performed for all of the PCR protocols described with the exception of the bfp and E-hly PCR, which needed an extension step of 7 min.
b
Primer mixture composed of equal amounts of oligonucleotide cdtS1 (consisting of bases not written in parentheses) and oligonucleotide cdtS2 (using bases in parentheses instead of the preceding ones).
c
Beginning with cycle 11, the extension time was prolonged for 20 s each cycle.