ABSTRACT
Helicobacter pylori [H. Pylori] is a human bacterial pathogen capable of surviving in the hostile environment of the stomach and duodenum. The bacterium has been linked to a number of gastric and extra gastric diseases. Recent reports of gastric infections caused by helicobacters other than H. Pylori created a clinical and research significance for a rapid, sensitive and specific diagnostic method. This is particularly important to make the laboratory-supported clinical diagnosis and follow up easier and specific. In this study, we used a multiplex PCR for the simultaneous detection and identification of Helicobacter genus and Helicobacter pylon in gastric fluid from patients with chronic non-ulcer dyspepsia. Patients with chronic dyspepsia of more than one month duration were recruited through the outpatient clinic in the university of Tanta hospital-Egypt for upper gastrointestinal endoscopy, only patients with no ulcer were involved in this study. Gastric fluid was obtained from 40 patients at the end of diagnostic upper gastrointestinal endoscopy, peripheral blood samples were also obtained and used to prepare serum for serodiagnosis. Gastric fluid was used for DNA preparation and inoculation of Skirrow's medium. Isolated pure colonies of H. pylori were used to prepare bacterial DNA to be used as positive control. Two pairs of primers; Hcom1, Hconn2 specific for Helicobacter genus, and Hicd1, Hicd2 specific for H. pylori species were used in the multiplex PCR. Two fragments of PCR products of 389 bp and 1200 bp were obtained from 34 specimen [85%] using Hcom1-Hcom2 and Hicd1-Hicd2 primers respectively. In five specimens [12.5%] a single band corresponding to the genus-specific gene and not the species-specific gene was obtained. In one specimen [2.5%], no DNA amplification was obtained. No DNA amplification of the negative control Gram-positive or Gram-negative bacteria was detected. The detection limit of the assay used in this study was 0.04 pg of DNA. The results obtained from this study demonstrate that 85% of the symptomatic gastric helicobacter infections are due to helicobacter pylori. They also demonstrate that this used protocol is a rapid, specific and sensitive assay for simultaneous detection of Helicobacter genus members and Helicobacter pylori species in gastric juice samples. This protocol reduces the number of PCR amplifications needed for specific diagnosis of the helicobacter infections. This can help physicians to have accurate and rapid diagnosis to cases of non ulcer dyspepsia, so that the right treatment can be precisely planned