Helicobacter pylori strain genotyping based on the amplification and sequencing of cagA gene

Abstract

Abstract Relevance: H. pylori presence has been described as a high risk factor of suffering gastric cancer, which is the fourth most common cancer in Colombia. Some studies [1, 2]; point out that when a genotypic characterization of different strains of this microorganism is carried out, more than one genotype can be found in the same patient can be found. Purpose: To characterize different H.pylori cagA genotypes that occur in the same patient sample (biopsy) applying molecular and bioinformatic techniques. Methods: cagA positive strains (n=35) from Instituto Nacional de Cancerología (Bogotá-Colombia), bank s samples were included as study material. Tissue DNA was extracted and used to amplify the 3’ region of the cagA gene by PCR [3]. The NTC11637 H. pylori strain was included as a positive control. PCR products were visualized in 2% agarose gels and the amplified fragments were purified using a commercial kit. As a final stage, a precipitation based on ethanol and EDTA was done and reaction products were sequenced. The obtained sequences were edited and submitted to a bioinformatic analysis using AASA, a program designed to characterize the EPIYA phosphorylation motifs localized in the C-terminal of the CagA protein by Rothstain, Yañez and Vivas (Los Andes University Thesis, 2008). Results: The molecular and bioinformatic approach described allowed strain characterization. The applied strategy leads to verify the presence of the A, B and C EPIYA motifs in different combinations. ABC group was the most frequent motif found (48%). These results match those of previous reports in Colombia, nevertheless it is important to remark that amplicons with different molecular sizes can have the same EPIYA motif and that amplicons with similar sizes can have different EPIYA motifs. Conclusions: The current strategy demonstrated to be useful in the identification and posterior characterization of more than one genotype of the different H. pylori strains present in the same biopsy sample. This molecular and bioinformatics approach combined with other techniques seems to be a good alternative to establish if an infection by H. pylori is the result of a coinfection or a microevolution process.