Detection of babA2

The presence of babA2 was verified by mismatch PCR using the following oligonucleotides: F5´-AATCCAAAAAGGAGAAAAAACATGAAA-3′ and R5′-TGTTAGTGATTTCGGTGTAGGACA-3′, designed by Gerhard et al. [16], (Fig. 2). The amplification reaction was performed in a final volume of 15 μL, with 3.0 mM MgCl₂, 0.25 mM dNTPs, 5 pmol of each oligonucleotide, 1 U of Taq DNA polymerase Platinum (Invitrogen, Carlsbad, CA, USA) and 600 ng of total DNA. The amplification program included an initial denaturation cycle at 95 °C for 3 min, 40 cycles at 95 °C for 30 s, 57 °C for 40 s, 72 °C for 45 s, and a final extension cycle at 72 °C for 5 min. The PCR products were subjected to electrophoresis on agarose gel (1.0%); the gels were stained with ethidium bromide and visualized under ultraviolet light (UV). The samples were considered babA2-positive when a band of 850 bp was observed.

Forward primer binding to babA2 and babA1 gene sequences. Detection of bab2A gene is determined by perfect match of 3′- end from forward primer. Differences between babA2 and bab1 sequences are shown in the increased font size and matches are indicated by vertical bars; mismatches are indicated by asterisks. babA1 and babA2 have almost complete sequence homology, with the exception of an approximately 10 bp insert, found only in babA2, which creates a translational initiation codon in the signal peptide sequence. Gerhard et al. used this sequence difference to amplify the babA2 gene selectively by mismatch PCR [16]

DNA from the ATCC 43504 strain of H. pylori (vacA s1m1/cagA+/babA2+) was used as positive control in all PCR reactions; template DNA was substituted by sterile deionized water as negative control. DNA from a gastric biopsy was used as positive control for s2 and m2 allele types. All the PCR reactions were performed in a Mastercycler Ep gradient thermal cycler (Eppendorf, Germany).