Table 1 Set of primers and PCR circumstances applied for genotyping of vacA, cagA, iceA, oipA and babA alleles
Genes | Primer Sequence (5′-3′) | Size of product (bp) | Volume of PCR reaction (50 μl) | PCR programs | |
|---|---|---|---|---|---|
VacA s1a | F: CTCTCGCTTTAGTAGGAGC R: CTGCTTGAATGCGCCAAAC | 213 | 5 μL PCR buffer 10 x 1.5 mM Mgcl2 200 μM dNTP (Thermo Fisher Scientific, St. Leon-Rot, Germany) 0.5 μM of each primers F & R 1.25 U Taq DNA polymerase (Thermo Fisher Scientific, St. Leon-Rot, Germany) 2.5 μL DNA template | 1 cycle: 95 °C ------------ 1 min. 32 cycle: 95 °C ------------ 45 s 64 °C ------------ 50 s 72 °C ------------ 70 s 1 cycle: 72 °C ------------ 5 min | |
VacA s1b | F: AGCGCCATACCGCAAGAG CTGCTTGAATGCGCCAAAC | 187 | |||
VacA s1c | F: CTCTCGCTTTAGTGGGGYT R: CTGCTTGAATGCGCCAAAC | 213 | |||
VacA s2 | F: GCTAACACGCCAAATGATCC R: CTGCTTGAATGCGCCAAAC | 199 | |||
VacA m1a | F: GGTCAAAATGCGGTCATGG R: CCATTGGTACCTGTAGAAAC | 290 | |||
VacA m1b | F: GGCCCCAATGCAGTCATGGA R: GCTGTTAGTGCCTAAAGAAGCAT | 291 | |||
VacA m2 | F: GGAGCCCCAGGAAACATTG R: CATAACTAGCGCCTTGCA | 352 | |||
Cag A | F: GATAACAGCCAAGCTTTTGAGG R: CTGCAAAAGATTGTTTGGCAGA | 300 | 5 μL PCR buffer 10X 2 mM Mgcl2 150 μM dNTP (Thermo Fisher Scientific, St. Leon-Rot, Germany) 0.75 μM of each primers F & R 1.5 U Taq DNA polymerase (Thermo Fisher Scientific, St. Leon-Rot, Germany) 3 μL DNA template | 1 cycle: 94 °C ------------ 1 min. 32 cycle: 95 °C ------------ 60 s 56 °C ------------ 60 s 72 °C ------------ 60 s 1 cycle: 72 °C ------------ 10 min | |
IceA | IceA1 | F: GTGTTTTTAACCAAAGTATC R: CTATAGCCASTYTCTTTGCA | 247 | 5 μL PCR buffer 10 x 2 mM Mgcl2 150 μM dNTP (Thermo Fisher Scientific, St. Leon-Rot, Germany) 0.75 μM of each primers F & R 1.5 U Taq DNA polymerase (Thermo Fisher Scientific, St. Leon-Rot, Germany) 3 μL DNA template | 1 cycle: 94 °C ------------ 1 min. 32 cycle: 94 °C ------------ 60 s 56 °C ------------ 60 s 72 °C ------------ 60 s 1 cycle: 72 °C ------------ 10 min |
IceA2 | F: GTTGGGTATATCACAATTTAT R: TTRCCCTATTTTCTAGTAGGT | 229/334 | |||
OipA | F: GTTTTTGATGCATGGGATTT R: GTGCATCTCTTATGGCTTT | 401 | 5 μL PCR buffer 10 x 2 mM Mgcl2 150 μM dNTP (Thermo Fisher Scientific, St. Leon-Rot, Germany) 0.75 μM of each primers F & R 1.5 U Taq DNA polymerase (Thermo Fisher Scientific, St. Leon-Rot, Germany) 3 μL DNA template | 1 cycle: 94 °C ------------ 1 min. 32 cycle: 94 °C ------------ 60 s 56 °C ------------ 60 s 72 °C ------------ 60 s 1 cycle: 72 °C ------------ 10 min | |
BabA | F: CCAAACGAAACAAAAAGCGT R: GCTTGTGTAAAAGCCGTCGT | 105–124 | 5 μL PCR buffer 10 x 2 mM Mgcl2 150 μM dNTP (Thermo Fisher Scientific, St. Leon-Rot, Germany) 0.75 μM of each primers F & R 1.5 U Taq DNA polymerase (Thermo Fisher Scientific, St. Leon-Rot, Germany) 3 μL DNA template | 1 cycle: 94 °C ------------ 1 min. 35 cycle: 94 °C ------------ 60 s 57 °C ------------ 45 s 72 °C ------------ 30 s 1 cycle: 72 °C ------------ 10 min | |