TABLE 2.
PCR primers and conditions for multilocus sequence analysis
| Primer designationa | Sequence (5′-3′) | Cycling conditions | Name of target gene | PCR product length (bp) | Length of sequence used (bp) |
|---|---|---|---|---|---|
| adk3 | GGG GAA AGG GAC TCA GGC TCA G | 94°C, 30 s; 57°C, 30 s; 72°C, 60 s | Adenylate kinase gene (adk) | 556 | 462 |
| adk4 | AAC TTT CGC GTA TTT GGT ATT | ||||
| arcA-p1 | GAA GAC GAG TTG GTA ACA CG | 94°C, 30 s; 58°C, 30 s; 72°C, 60 s | Gene for respiration control protein ArcA (arcA) | 645 | 544 |
| arcA-p2NW | CTT CCA GAT CAC CGC AGA AGC | ||||
| fumC-1 | CGG CTC CGG CAC GCA AAG TAA | 94°C, 30 s; 60°C, 60 s; 72°C, 60 s | Fumarate dehydrogenase gene (fumC) | 753 | 514 |
| fumC-2 | ATC GCC ACG TCG TTC CCC ATC A | ||||
| icd+39 | CGG CAC AAG GCA AGA AG | 94°C, 30 s; 49°C, 30 s; 72°C, 60 s | Isocitrate dehydrogenase gene (icdA) | 630 | 542 |
| icd−669 | GCA CCA GAG TCA CAG AG | ||||
| mlp-p3 | CGT TGA TCT GAT TGC TCA GG | 94°C, 30 s; 55°C, 60 s; 72°C, 120 s | Mannitol-1-phosphate dehydrogenase gene (mtlD) | 580 | 410 |
| mlp-p4 | TGA TCA GTA CCG CAC CAC TT | ||||
| mdh + 55 | CTG TTA AAA ACC CAA CTG CC | 94°C, 30 s; 52°C, 30 s; 72°C, 60 s | Malate dehydrogenase gene (mdh) | 817 | 496 |
| mdh−872 | GCG TTC TGT TCA AAT GCG | ||||
| pgi-p2 | GGC GGT TAC CTT CGA ATA CT | 94°C, 30 s; 49°C, 50 s; 72°C, 60 s | Phosphoglucose isomerase gene (pgi) | 730 | 581 |
| pgi-p3 | GCGTGA CTG GTT CCT GA | ||||
| SK1 | CCC GAA TTC GGC ACA AGC ATA AGC | 94°C, 30 s; 52°C, 60 s; 72°C, 60 s | Intimin gene (eae) | 863 | 725 |
| SK2 | CCC GGA TCC GTC TCG CCA GTA TTC G | ||||
| escD+164 | CTT GAT ATA CGT GAG GAA GG | 94°C, 30 s; 52°C, 30 s; 72°C, 60 s | E. coli secreted protein gene D (escD) | 673 | 587 |
| escD−837 | CTC TGA TGC CAG TTC ATT G |
a
MLST PCR primers for housekeeping were designed on the basis of the sequence of E. coli K-12 (accession no. NC_000913) and were similar to a scheme published by T. Whittam, Michigan State University, East Lansing, Mich. (http://www.shigatox.net/stec/mlst-new/index.html). SK1 and SK2 were published earlier (51), and escD + 164 and −837 were complementary to sequences of the LEE of EPEC E2348/69 (24).