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Table 4 PCR primers and conditions for amplification of STEC virulence genes and eae typing

From: Serotypes, virulence genes and intimin types of Shiga toxin (verocytotoxin)-producing Escherichia coli isolates from minced beef in Lugo (Spain) from 1995 through 2003

Gene

Primer

Oligonucleotide sequence (5'-3')

Fragment size (bp)

Annealing temperature

Primer Coordinatesa

Accession number

stx1

VT1-A

CGCTGAATGTCATTCGCTCTGC

302

55°C

113–134

M17358

 

VT1-B

CGTGGTATAGCTACTGTCACC

  

394–414

 

stx 2

VT2-A

CTTCGGTATCCTATTCCCGG

516

55°C

50–69

M59432

 

VT2-B

CTGCTGTGACAGTGACAAAACGC

  

543–565

 

ehxA

HlyA1

GGTGCAGCAGAAAAAGTTGTAG

1.551

60°C

238–259

X79839

 

HlyA4

TCTCGCCTGATAGTGTTTGGTA

  

1767–1788

 

eae b

EAE-1

GGAACGGCAGAGGTTAATCTGCAG

346

55°C

631–654

AF022236

 

EAE-2

GGCGCTCATCATAGTCTTTC

  

957–976

 

eae-α1

EAE-FB

AAAACCGCGGAGATGACTTC

820

60°C

1909–1928

AF022236

 

EAE-A

CACTCTTCGCATCTTGAGCT

  

2709–2728

 

eae-α2

IH2498aF

AGACCTTAGGTACATTAAGTAAGC

517

60°C

2099–2122

AF530555

 

IH2498aR

TCCTGAGAAGAGGGTAATC

  

2597–2615

 

eae-β1

B1F

CACAATTAATGCACCGGGT

241

55°C

2499–2517

AF453441

 

B1R

GCTTGATACACCTGATGACT

  

2720–2739

 

eae-ξR/β2B

B2F

TGAAGGGGGGAACCCCTGTG

564

62°C

2054–2073

AF530556

 

B2R

TTTCTTTTGACTGTGCTAAAGC

  

2596–2617

AJ715407

eae-δ/κ/β2O

EAE-FB

AAAACCGCGGAGATGACTTC

830

60°C

1909–1928

U66102

 

EAE-D

CTTGATACACCCGATGGTAAC

  

2718–2738

 

eae-γ1

EAE-FB

AAAACCGCGGAGATGACTTC

804

60°C

1909–1928

AF071034

 

EAE-C1

AGAACGCTGCTCACTAGATGTC

  

2691–2712

 

eae-θ/γ2

EAE-C2F

AGAACGTTACTGGTGACTTA

414

58°C

2303–2322

AF025311

 

EAE-C2R

CTGATATTTTATCAGCTTCA

  

2697–2716

 

eae-ε1

EAE-FB

AAAACCGCGGAGATGACTTC

722

66°C

1909–1928

AF116899

 

LP5

AGCTCACTCGTAGATGACGGCAAGCG

  

2605–2630

 

eae-νR/ε2

EAE-E2F

AATACAGAAGTTAAGGCAT

378

58°C

2230–2248

AF530554

 

EAE-E2R

ACGACCACTATTCATTTC

  

2590–2607

 

eae

Z1

GGTAAGCCGTTATCTGCC

206

62°C

2062–2079

AF449417

 

Z2

ATAGCAAGTGGGGTGAAG

  

2250–2267

 

eae-η1/η2

EAE-FB

AAAACCGCGGAGATGACTTC

702

60°C

1899–1918

AJ308550

 

ETA-B

TAAGCGACCACTATTCGTG

  

2582–2600

 

eae-η1

ETA-FN

CGCTTTGTTTAATGCCGATAAA

410

62°C

1074–1095

AJ308550

 

ETA-RN

GACTGCGTAATGCACTG

  

1467–1483

 

eae-ι1

EAE-FB

AAAACCGCGGAGATGACTTC

651

55°C

1909–1928

AJ308551

 

IOTA-B

GTCATATTTAACTTTTACACTA

  

2538–2559

 

eae-μR/ι2,

Iota2-F

CTGGTAAAGCGATAGTCAAAC

936

58°C

1850–1870

AF530553

 

Iota2-R

GCGTTTTTGAAGAAACATTTTGC

  

2763–2785

 

eae

68.4F

CGGTCAGCCTGTGAAGGGC

370

64°C

2061–2079

AJ715409

 

68.4R

AATACCGGAAGAGGCATCTAT

  

2410–2430

 

eae-μB

EAE-FB

AAAACCGCGGAGATGACTTC

655

60°C

1909–1928

AJ705049

 

FV373R

ACTCATCATAATAAGCTTTTTGG

  

2541–2563

 

eae-νB

IH1229aF

CACAGCTTACAATTGATAACA

311

60°C

2255–2275

AJ705050

 

IH1229aR

CTCACTATAAGTCATACGACT

  

2545–2565

 

eae-ξB

EAE-FB

AAAACCGCGGAGATGACTTC

468

66°C

1909–1928

AJ705051

 

B49R

ACCACCTTTAGCAGTCAATTTG

  

2355–2376

 

eae-ο

H2997fF

AGCGTTAGCAATGCCGCAGTTGAT

271

60°C

2203–2226

AJ876647

 

IH2997fR

CAACGGTAATTGTTGTTTCC

  

2454–2473

AJ876648

  1. a Location within eae gene. The oligonucleotide primers were designed by us according to the nucleotide sequences of the virulence genes.
  2. bUniversal oligonucleotide primer pair EAE1 and EAE-2 with homology to the 5'conserved region of eae gene (detects all types of eae variants described at the moment). Isolates positive for eae gene with EAE-1 and EAE-2 primers were further analysed with all different variant primers.