Table 6 Oligonucleotide primers and PCR conditions used for genotyping of Helicobacter pylori strains isolated from Iranian bottled mineral water
From: Helicobacter pylori in bottled mineral water: genotyping and antimicrobial resistance properties
Genes | Primer Sequence (5’-3’) | Size of product (bp) | Volume of PCR reaction (50 μl) | PCR programs |
|---|---|---|---|---|
vacA s 1 a | F: CTCTCGCTTTAGTAGGAGC | 213 | 5 μL PCR buffer 10X | 1 cycle: 95 °C ------------ 1 min. |
R: CTGCTTGAATGCGCCAAAC | ||||
vacA s 1 b | F: AGCGCCATACCGCAAGAG | 187 | 1.5 mM Mgcl2 | 32 cycle: 95 °C ------------ 45 s |
CTGCTTGAATGCGCCAAAC | 200 μM dNTP (Fermentas) | |||
vacA s 1 c | F: CTCTCGCTTTAGTGGGGYT | 213 | 64 °C ------------ 50 s | |
R: CTGCTTGAATGCGCCAAAC | 0.5 μM of each primers F & R | |||
vacA s 2 | F: GCTAACACGCCAAATGATCC | 199 | 72 °C ------------ 70 s | |
R: CTGCTTGAATGCGCCAAAC | 1.25 U Taq DNA polymerase (Fermentas) | |||
vacA m 1 A | F: GGTCAAAATGCGGTCATGG | 290 | 1 cycle: 72 °C ------------ 5 min | |
R: CCATTGGTACCTGTAGAAAC | ||||
vacA m 1 B | F: GGCCCCAATGCAGTCATGGA | 291 | 2.5 μL DNA template | |
R: GCTGTTAGTGCCTAAAGAAGCAT | ||||
vacA m 2 | F: GGAGCCCCAGGAAACATTG | 352 | ||
R: CATAACTAGCGCCTTGCA | ||||
cag A | F: GATAACAGCCAAGCTTTTGAGG | 300 | 5 μL PCR buffer 10X | 1 cycle: 94 °C ------------ 1 min. |
R: CTGCAAAAGATTGTTTGGCAGA | ||||
2 mM Mgcl2 | 32 cycle: 95 °C ------------ 60 s | |||
150 μM dNTP (Fermentas) | ||||
0.75 μM of each primers F & R | 56 °C ------------ 60 s | |||
1.5 U Taq DNA polymerase (Fermentas) | 72 °C ------------ 60 s | |||
1 cycle: 72 °C ------------ 10 min | ||||
3 μL DNA template | ||||
iceA 1 | F: GTGTTTTTAACCAAAGTATC | 247 | 5 μL PCR buffer 10X | 1 cycle: 94 °C ------------ 1 min. |
R: CTATAGCCASTYTCTTTGCA | ||||
iceA 2 | F: GTTGGGTATATCACAATTTAT | 229/334 | 2 mM Mgcl2 | 32 cycle: 94 °C ------------ 60 s |
R: TTRCCCTATTTTCTAGTAGGT | 200 μM dNTP (Fermentas) | |||
56 °C ------------ 60 s | ||||
0.5 μM of each primers F & R | ||||
72 °C ------------ 60 s | ||||
1.5 U Taq DNA polymerase (Fermentas) | ||||
1 cycle: 72 °C ------------ 8 min | ||||
5 μL DNA template | ||||
oip A | F: GTTTTTGATGCATGGGATTT | 401 | 5 μL PCR buffer 10X | 1 cycle: 94 °C ------------ 2 min. |
R: GTGCATCTCTTATGGCTTT | ||||
2.5 mM Mgcl2 | 32 cycle: 94 °C ------------ 60 s | |||
200 μM dNTP (Fermentas) | ||||
56 °C ------------ 60 s | ||||
0.5 μM of each primers F & R | ||||
72 °C ------------ 60 s | ||||
2 U Taq DNA polymerase (Fermentas) | ||||
1 cycle: 72 °C ------------ 10 min | ||||
3 μL DNA template | ||||
cagE | F: TTGAAAACTTCAAGGATAGGATAGAGC R: GCCTAGCGTAATATCACCATTACCC | 508 | 5 μL PCR buffer 10X | 1 cycle: 94 °C ------------ 1 min. |
2 mM Mgcl2 | 35 cycle: 94 °C ------------ 60 s | |||
150 μM dNTP (Fermentas) | ||||
53 °C ------------ 45 s | ||||
0.75 μM of each primers F & R | ||||
72 °C ------------ 45 min | ||||
1.5 U Taq DNA polymerase (Fermentas) | ||||
1 cycle: 72 °C ------------ 8 min | ||||
3 μL DNA template | ||||
BabA2 | F: CCAAACGAAACAAAAAGCGT | 271 | 5 μL PCR buffer 10X | 1 cycle: 95 °C ------------ 1 min. |
R: GCTTGTGTAAAAGCCGTCGT | ||||
1.5 mM Mgcl2 | 30 cycle: 91 °C ------------ 60 s | |||
200 μM dNTP (Fermentas) | ||||
45 °C ------------ 60 s | ||||
0.5 μM of each primers F & R | ||||
72 °C ------------ 60 s | ||||
1.25 U Taq DNA polymerase (Fermentas) | ||||
1 cycle: 72 °C ------------ 8 min | ||||
2.5 μL DNA template |