TABLE 1.
PCR primer and conditions for amplification of STEC virulence genes
| Gene | Primers | Oligonucleotide sequence (5′-3′)c | Fragment size (bp) | Annealing temp (°C) | Primer coordinates | GenBank accession no. |
|---|---|---|---|---|---|---|
| stx1 | VT1-A | CGCTGAATGTCATTCGCTCTGC | 302 | 55 | 113-134 | M17358 |
| VT1-B | CGTGGTATAGCTACTGTCACC | 394-414 | ||||
| stx2 | VT2-A | CTTCGGTATCCTATTCCCGG | 516 | 55 | 50-69 | M59432 |
| VT2-B | CTGCTGTGACAGTGACAAAACGC | 543-565 | ||||
| ehxA | HlyA1 | GGTGCAGCAGAAAAAGTTGTAG | 1,551 | 60 | 238-259 | X79839 |
| HlyA4 | TCTCGCCTGATAGTGTTTGGTA | 1767-1788 | ||||
| eaea | EAE-1 | GGAACGGCAGAGGTTAATCTGCAG | 775 | 55 | 1441-1460 | AF022236 |
| EAE-2 | GGCGCTCATCATAGTCTTTC | 2193-2215 | ||||
| eaeb | EAE-F | ATTACTGAGATTAAGGCTGAT | 682 | 55 | 1978-1998 | AF022236 |
| EAE-RB | ATTTATTTGCAGCCCCCCAT | 2640-2659 | ||||
| eae-α1 | EAE-FB | AAAACCGCGGAGATGACTTC | 820 | 60 | 1909-1928 | AF022236 |
| EAE-A | CACTCTTCGCATCTTGAGCT | 2709-2728 | ||||
| eae-α2 | IH2498aF | AGACCTTAGGTACATTAAGTAAGC | 517 | 60 | 2099-2122 | AF530555 |
| IH2498aR | TCCTGAGAAGAGGGTAATC | 2597-2615 | ||||
| eae-β | EAE-FB | AAAACCGCGGAGATGACTTC | 830 | 64 | 1909-1928 | AF453441 |
| EAE-B | CTTGATACACTTGATGACTGT | 2718-2738 | ||||
| eae-β1 | EA-B1-F | CGCCACTTAATGCCAGCG | 811 | 60 | 1928-1945 | AF453441 |
| EAE-B | CTTGATACACCTGATGACTGT | 2718-2738 | ||||
| eae-β2 | EA-B2-F | CCCGCCACTTAATCGCACGT | 807 | 60 | 1929-1948 | AF043226 |
| EAE-B | CTTGATACACCTGATGACTGT | 2715-2735 | ||||
| eae-γ1 | EAE-FB | AAAACCGCGGAGATGACTTC | 804 | 60 | 1909-1928 | AF071034 |
| EAE-C1 | AGAACGCTGCTCACTAGATGTC | 2691-2712 | ||||
| eae-γ2/θ | EAE-FB | AAAACCGCGGAGATGACTTC | 808 | 58 | 1909-1928 | AF025311 |
| EAE-C2 | CTGATATTTTATCAGCTTCA | 2697-2716 | ||||
| eae-δ/κ | EAE-FB | AAAACCGCGGAGATGACTTC | 833 | 60 | 1909-1928 | U66102 |
| EAE-D | CTTGATACACCCGATGGTAAC | 2721-2741 | ||||
| eae-ɛ | EAE-FB | AAAACCGCGGAGATGACTTC | 722 | 66 | 1909-1928 | AF116899 |
| LP5 | AGCTCACTCGTAGATGACGGCAAGCG | 2605-2630 | ||||
| eae-ζ | Z1 | GGTAAGCCGTTATCTGCC | 206 | 62 | 2062-2079 | AF449417 |
| Z2 | ATAGCAAGTGGGGTGAAG | 2250-2267 | ||||
| eae-η | EAE-FB | AAAACCGCGGAGATGACTTC | 712 | 62 | 1899-1918 | AJ308550 |
| LP8 | TAGATGACGGTAAGCGAC | 2593-2610 | ||||
| eae-ι | EAE-FB | AAAACCGCGGAGATGACTTC | 807 | 66 | 1909-1928 | AJ308551 |
| LP7 | TTTATCCTGCTCCGTTTGCT | 2695-2715 | ||||
| eae-λ | 68.4F | CGGTCAGCCTGTGAAGGGC | 466 | 64 | 2061-2079 | AF530557 |
| 68.4R | ATAGATGCCTCTTCCGGTATT | 2506-2526 | ||||
| eae-μ | FV373F | CAACGGTAAGTCTCAGACAC | 443 | 64 | 114-133 | AJ579305 |
| FV373R | CATAATAAGCTTTTTGGCCTACC | 534-556 | ||||
| eae-ν | IH1229aF | CACAGCTTACAATTGATAACA | 311 | 60 | 269-289 | AJ579306 |
| IH1229aR | CTCACTATAAGTCATACGACT | 559-579 | ||||
| eae-ξ | EAE-FB | AAAACCGCGGAGATGACTTC | 468 | 66 | 1909-1928 | AF116899 |
| B49R | ACCACCTTTAGCAGTCAATTTG | 363-384 | AJ582912 |
a
Universal oligonucleotide primer pair EAE-1 and EAE-2 with homology to the 5′ conseved region of eae (detects all types of eae variants described at the moment).
b
Universal oligonucleotide primer pair EAE-F and EAE-RB with homology to the 3′ variable region of eae (detects all types of eae variants described at the moment).
c
HlyA1 and Hly4 primer pair was designed by Schmidt et al. (35). The remaining primer pairs were designed by us according to the nucleotide sequences of the genes (10; Blanco et al., submitted).