Table 8 PCR programs (temperature and volume) for detection of STEC O-serogroups, virulence factors and antibiotic resistance genes

O157, O145, O103, O26, O111

1 cycle:

95°C ------------ 3 min.

5 μL PCR buffer 10X

30 cycle:

1.5 mM Mgcl₂

95°C ------------ 20 s

200 μM dNTP^e

58°C ------------ 40 s

0.5 μM of each primers F & R

72°C ------------ 30 s

1.25 U Taq DNA polymerase^e

1 cycle:

2.5 μL DNA template

72°C ------------ 8 min

O91, O128, O121, O113, O45

1 cycle:

94°C ------------ 6 min.

5 μL PCR buffer 10X

34 cycle:

2 mM Mgcl₂

95°C ------------ 50 s

150 μM dNTP

58°C ------------ 70 s

0.75 μM of each primers F & R

72°C ------------ 55 s

1.5 U Taq DNA polymerase

1 cycle:

3 μL DNA template

72°C ------------ 10 min

LT, ST

1 cycle:

94°C ------------ 6 min.

5 μL PCR buffer 10X

34 cycle:

1.5 mM Mgcl₂

95°C ------------ 50 s

200 μM dNTP

56°C ------------ 70 s

0.5 μM of each primers F & R

72°C ------------ 50 s

1 U Taq DNA polymerase

1 cycle:

4 μL DNA template

72°C ------------ 6 min

stx1, stx2, eae, hly, cnf1, cnf2, cdtIII, f17c

1 cycle:

95°C ------------ 3 min.

5 μL PCR buffer 10X

34 cycle:

2 mM Mgcl₂

94°C ------------ 60 s

200 μM dNTP

56°C ------------ 45 s

0.5 μM of each primers F & R

72°C ------------ 60 s

1.5 U Taq DNA polymerase

1 cycle:

5 μL DNA template

72°C ------------ 10 min

aadA1, tetA, tetB, dfrA1, qnr, Sul1, aac[3]-IV, blaSHV, CITM, cat1, cmlA

1 cycle:

94°C ------------ 8 min.

5 μL PCR buffer 10X

32 cycle:

2.5 mM Mgcl₂

95°C ------------ 60 s

200 μM dNTP

55°C ------------ 70 s

0.5 μM of each primers F & R

72°C ------------ 2 min

2 U Taq DNA polymerase

1 cycle:

3 μL DNA template

72°C ------------ 8 min