Table 1 Primers and PCR conditions used for characterization of virulence genes, O-serogroups and antibiotic resistance genes in the STEC strains isolated from raw milk and traditional dairy product samples
| Target gene | Primer sequence (5′-3′) | PCR product (bp) | PCR programs | PCR Volume (50 μL) |
|---|---|---|---|---|
| O157 |
F: CGGACATCCATGTGATATGG R: TTGCCTATGTACAGCTAATCC | 259 |
1 cycle: 95 0C ------------ 3 min. |
5 μL PCR buffer 10X 2 mM Mgcl2 |
| O145 |
F: CCATCAACAGATTTAGGAGTG R: TTTCTACCGCGAATCTATC | 609 |
30 cycle: 95 0C ------------ 20 s 58 0C ------------ 40 s 72 0C ------------ 30 s |
150 μM dNTP (Fermentas) 0.75 μM of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) |
| O103 |
F: TTGGAGCGTTAACTGGACCT R: GCTCCCGAGCACGTATAAG | 321 | ||
| O26 |
F: CAGAATGGTTATGCTACTGT R: CTTACATTTGTTTTCGGCATC | 423 | ||
| O111 |
F: TAGAGAAATTATCAAGTTAGTTCC R: ATAGTTATGAACATCTTGTTTAGC | 406 |
1 cycle: 72 0C ------------ 8 min | 3 μL DNA template |
| O91 |
F: GCTGACCTTCATGATCTGTTGA R: TAATTTAACCCGTAGAATCGCTGC | 291 |
1 cycle: 94 0C ------------ 6 min. 34 cycle: 95 0C ------------ 50 s 58 0C ------------ 70 s 72 0C ------------ 55 s 1 cycle: 72 0C ------------ 10 min |
5 μL PCR buffer 10X 2 mM Mgcl2 150 μM dNTP (Fermentas) 0.75 μM of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) 3 μL DNA template |
| O128 |
F: GCTTTCTGCCGATATTTGGC R: CCGACGGACTGATGCCGGTGATT | 289 | ||
| O121 |
F: TGGCTAGTGGCATTCTGATG R: TGATACTTTAGCCGCCCTTG | 322 | ||
| O113 |
F: GGGTTAGATGGAGCGCTATTGAGA R: AGGTCACCCTCTGAATTATGGCAG | 771 | ||
| O45 |
F: CCGGGTTTCGATTTGTGAAGGTTG R: CACAACAGCCACTACTAGGCAGAA | 527 | ||
| stx1 |
F: AAATCGCCATTCGTTGACTACTTCT R: TGCCATTCTGGCAACTCGCGATGCA | 366 |
1 cycle: 95 0C ------------ 3 min. 34 cycle: 94 0C ------------ 60 s 56 0C ------------ 45 s 72 0C ------------ 60 s 1 cycle: 72 0C ------------ 10 min |
5 μL PCR buffer 10X 2 mM Mgcl2 150 μM dNTP (Fermentas) 0.75 μM of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) 3 μL DNA template |
| stx2 |
F: CGATCGTCACTCACTGGTTTCATCA R: GGATATTCTCCCCACTCTGACACC | 282 | ||
| eaeA |
F: TGCGGCACAACAGGCGGCGA R: CGGTCGCCGCACCAGGATTC | 629 | ||
| ehly |
F: CAATGCAGATGCAGATACCG R: CAGAGATGTCGTTGCAGCAG | 432 | ||
| aadA1 |
F: TATCCAGCTAAGCGCGAACT R: ATTTGCCGACTACCTTGGTC | 447 |
1 cycle: 94 0C ------------ 8 min. 32 cycle: 95 0C ------------ 60 s 55 0C ------------ 70 s 72 0C ------------ 2 min 1 cycle: 72 0C ------------ 8 min |
5 μL PCR buffer 10X 2 mM Mgcl2 150 μM dNTP (Fermentas) 0.75 μM of each primers F & R 1.5 U Taq DNA polymerase (Fermentas) 3 μL DNA template |
| tetA |
F: GGTTCACTCGAACGACGTCA R: CTGTCCGACAAGTTGCATGA | 577 | ||
| tetB |
F: CCTCAGCTTCTCAACGCGTG R: GCACCTTGCTGATGACTCTT | 634 | ||
| dfrA1 |
F: GGAGTGCCAAAGGTGAACAGC R: GAGGCGAAGTCTTGGGTAAAAAC | 367 | ||
| qnr |
F: GGGTATGGATATTATTGATAAAG R: CTAATCCGGCAGCACTATTTA | 670 | ||
| aac(3)-IV |
F: CTTCAGGATGGCAAGTTGGT R: TCATCTCGTTCTCCGCTCAT | 286 | ||
| sul1 |
F: TTCGGCATTCTGAATCTCAC R: ATGATCTAACCCTCGGTCTC | 822 | ||
| blaSHV |
F: TCGCCTGTGTATTATCTCCC R: CGCAGATAAATCACCACAATG | 768 | ||
| CITM |
F: TGGCCAGAACTGACAGGCAAA R: TTTCTCCTGAACGTGGCTGGC | 462 | ||
| cat1 |
F: AGTTGCTCAATGTACCTATAACC R: TTGTAATTCATTAAGCATTCTGCC | 547 | ||
| cmlA |
F: CCGCCACGGTGTTGTTGTTATC R: CACCTTGCCTGCCCATCATTAG | 698 |