Molecular identification of Helicobacter DNA in human gastric adenocarcinoma tissues using Helicobacter species-specific 16S rRNA PCR amplification and pyrosequencing analysis

Abstract

Helicobacter pylori (H. pylori) is a microaerophilic gram-negative bacterium known to be associated with chronic gastritis, peptic ulcer and gastric adenocarcinoma. In the present study, the presence of Helicobacter DNA was investigated using a Helicobacter species-specific 16S rRNA PCR amplification and pyrosequencing analysis in 51 resected gastric adenocarcinomas. DNA was extracted from paraffin-embedded tissues of resected gastric adenocarcinomas. PCR primers were designed to amplify the 133-bp PCR fragment in highly conserved regions of the 16S rRNA gene. The sequence of the PCR products was analyzed using a PSQ 96 system with SQA software. The pyrosequencing analysis of 16S rRNA showed that H. pylori was present in 47 (92.2%) of the 51 gastric adenocarcinomas. In the 4 H. pylori-negative cases, Helicobacter cinaedi (2 cases), Helicobacter mustelae (1 case) and Campylobacter hyointestinalis (1 case) were detected. Pyrosequencing technology was useful in the identification and differentiation of H. pylori from other species by analyzing the gene encoding 16S rRNA. Gastric adenocarcinoma tissues contain bacteria, and the majority are H. pylori. Helicobacter cinaedi, Helicobacter mustelae and Campylobacter hyointestinalis rarely occur. The roles of these organisms in the pathogenesis of gastric adenocarcinoma remain unclear.