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.
Gene
PCR program
PCR volume (50 L)
O157, O145, O103, O26, O111
1 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, O45
1 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