Primers and polymerase chain reaction (PCR) conditions used for amplification of the different DNA targets
| Name | Sequence | Organism | Gene | Cycling conditions | Amplicon size (bp) | Reference |
|---|---|---|---|---|---|---|
| Cycling conditions: after four minutes of initial denaturation at 94°C, each reaction mixture was amplified for 35–40 cycles under the conditions indicated, depending on the PCR. After the last cycle, a final extension was continued for another seven minutes. | ||||||
| F, forward; R, reverse. | ||||||
| Partial Genbank available cdtB sequences of H pullorum (AF220065), H bilis (AF243077), and H hepaticus (AF163667 and AF243076) were aligned in order to design the most possible specific H pullorum and H bilis PCR on the cdtB gene. Forward F1- and F2-cdtB-pullorum were designed to be used with the R2-cdtB-pullorum reverse primer. | ||||||
| *All of these PCR were species specific, with control strains listed in materials and methods. F4/R3-cdtB primers were used to determine the nucleotide sequence of H pullorum cdtB gene. | ||||||
| HS-1 | AACGATGAAGCTTCTAGCTTGCTAG | Helicobacter genus | 16S rRNA | (94°C; 30 sec, 60°C; 1 min 72°C; 30 sec)×40 | 399 | Germani21 |
| HS-2 | GTGCTTATTCGTTAGATACCGTCAT | Campylobacter genus | Avenaud12 | |||
| C97 | GCTATGACGGGTATCC | Helicobacter genus | 16S rRNA | (94°C; 30 sec, 54°C; 90 sec 72°C; 60 sec)×35 | 398 | Fox20 |
| C98 | GATTTTACCCCTACACCA | |||||
| F2-16S-CHPEC | ATCCTGGCTCAGAGTGAACG | Pseudo-universal | 16S rRNA | (94°C; 30 sec, 60°C; 30 sec, 72°C; 80 sec)×40 | 1456 | This study |
| R4-16S-CHPEC | CCTACGGTTACCTTGTTACGAC | |||||
| C96 | ACGGGTGAGTAACGCATAGG | Helicobacter genus | 16S rRNA | (94°C; 30 sec, 60°C; 30 sec, 72°C; 80 sec)×40 | 1374 | This study |
| R4-16S-CHPEC | CCTACGGTTACCTTGTTACGAC | |||||
| F1-mdh-coli | TTGTCGAATGTGCCTACGTT | Escherichia coli* | mdh | (94°C; 1 min, 60°C; 1 min, 72°C; 1 min)×35 | 130 | This study |
| R1-mdh-coli | ATACCTTCCAGCGCGTTCT | |||||
| No name | ATGAATGCTAGTTGTTGTGAG | Helicobacter pullorum | 16S rRNA | (94°C; 30 sec, 60°C; 1 min, 72°C; 80 sec)×40 | 467 | Stanley24 |
| No name | GATTGGCTCCACTTCACA | Helicobacter canadensis | ||||
| HPY S | AGGTTAAGAGGATGCGTCAGTC | Helicobacter pylori* | 23S rRNA | (94°C; 1 min, 55°C; 1 min, 72°C; 1 min)×40 | 267 | Ménard33 |
| HPY A | CGCATGATATTCCCATTAGCAGT | |||||
| F1-glmM-HP | AACGCCCTTTCTTCTCAAGC | Helicobacter pylori* | glmM | (94°C; 30 sec, 60°C; 30 sec, 72°C; 10 sec)×40 | 130 | This study |
| R1-glmM-HP | GCATGCATTCGCTCACAA | |||||
| F1-cdtB-pullorum | GTCTTTTGAGTGGATTGGATTCT | Helicobacter pullorum* | cdtB | (94°C; 30 sec, 60°C; 1 min, 72°C; 20 sec)×40 | 140 | This study |
| F2-cdtB-pullorum | GCCAGCAAGTCTTTTGAGTG | 148 | ||||
| R2-cdtB-pullorum | CACTCCGGGTGCTTGTGTAT | |||||
| F4-cdtB | TCAGCTAGCACAGAAAATAAATGG | Non-specific Helicobacter pullorum | cdtB | (94°C; 30 sec, 55°C; 60 sec, 72°C; 30 sec)×40 | 719 | This study |
| R3-cdtB | ACTAGCTGCTGCCAAAATCG | |||||
| F2-cdtB-bilis | CGAATCTATTATCCGGGCTTG | Helicobacter bilis* | cdtB | (94°C; 30 sec, 60°C; 1 min, 72°C; 30 sec)×40 | 151 | This study |
| R2-cdtB-bilis | GCCAAGCGAGTTCTATCATTAG | |||||
| F1-ureB-rappini | GATGATTAGGGCGACACAGC | Helicobacter rappini* | ureB | (94°C; 30 sec, 60°C; 30 sec, 72°C; 10 sec)×40 | 101 | This study |
| R2-ureB-rappini | CCCCAGATTCTATCTGCTTACTC | |||||