Research Article

Shiga Toxin-Producing Escherichia coli Isolated from Bovine Mastitic Milk: Serogroups, Virulence Factors, and Antibiotic Resistance Properties

Table 4

PCR conditions for detection of serogroups, virulence genes and antimicrobial resistance genes in Shiga toxin-producing Escherichia coli in bovine mastitis.

GenePCR programPCR volume (50  L)

O157, O145, O103, O26, O1111 cycle:
95°C—3 min
30 cycle:
95°C—20 s
58°C—40 s
72°C—30 s
1 cycle:
72°C—8 min
5 μL PCR buffer 10X
1.5 mM MgCl2
200 μM dNTP (Fermentas)
0.5 μM of each primers F and R
1.25 U Taq DNA polymerase (Fermentas)
2.5 μL DNA template

O91, O128, O121, O113, O451 cycle:
94°C—6 min
34 cycle:
95°C—50 s
58°C—70 s
72°C—55 s
1 cycle:
72°C—10 min
5 μL PCR buffer 10X
2 mM MgCl2
150 μM dNTP (Fermentas)
0.75 μM of each primers F and R
1.5 U Taq DNA polymerase (Fermentas)
3 μL DNA template

stx1, stx2, eaeA, ehly 1 cycle:
95°C—3 min
34 cycle:
94°C—60 s
56°C—45 s
72°C—60 s
1 cycle:
72°C—10 min
5 μL PCR buffer 10X
2 mM MgCl2
200 μM dNTP (Fermentas)
0.5 μM of each primers F and R
1.5 U Taq DNA polymerase (Fermentas)
5 μL DNA template

aadA1, tetA, tetB, dfrA1, qnr, aac(3)-IV, sul1, blaSHV, CITM, cat1, cmlA 1 cycle:
94°C—8 min
32 cycle:
95°C—60 s
55°C—70 s
72°C—2 min
1 cycle:
72°C—8 min
5 μL PCR buffer 10X
2.5 mM MgCl2
200 μM dNTP (Fermentas)
0.5 μM of each primers F and R
2 U Taq DNA polymerase (Fermentas)
3 μL DNA template