Direct polymerase chain reaction test for detection of Helicobacter pylori in humans and animals

Abstract

We designed a polymerase chain reaction (PCR) for amplifying the Helicobacter pylori gene encoding 16S rRNA. Primers for the specific detection of H. pylori were designed for areas of the 16S rRNA gene in which there is the least sequence homology between H. pylori and its closest relatives. The specificity of detection was confirmed by ensuring that the primers did not amplify DNA extracts from the campylobacters H. cinaedi, H. mustelae, and Wolinela succinogenes, which are the closest relatives of H. pylori, as determined by 16S rRNA sequencing. Serial dilution experiments revealed the detection of as little as 0.1 pg of DNA by PCR and 0.01 pg by nested PCR. H. pylori DNA was detected successfully in clinical paraffin-embedded and fresh gastric biopsy specimens from patients positive for the bacterium and also in fecal suspensions seeded with the organism. The DNA from the nonculturable coccoid form of H. pylori was also identified by the primers. Universal primers designed for highly conserved areas on the 16S rRNA gene enabled large amplification products to be produced for direct sequencing analysis. Gastric bacteria resembling H. pylori have been isolated from animals. DNA of these animal gastric bacteria amplified with H. pylori-specific primers yielded PCR products identical to those from human isolates of H. pylori, as confirmed by the use of a 20-base radiolabelled probe complementary to an internal sequence flanked by the H. pylori-specific primers. The results of PCR amplification and partial 16S rRNA gene sequence analysis strongly support the contention that the gastric organisms previously recovered from a pig, a baboon, and rhesus monkeys are H. pylori.

Polymerase chain reaction based analysis of the cytotoxin associated gene pathogenicity island of Helicobacter pylori from saliva: An approach for rapid molecular genotyping in relation to disease status

“…Thus, a scrupulous analysis carried out in the present study to assess the status of the cag genes in ulcer patients and non-ulcer patients showed the prevalence of most of the genes of this intricate island in the DNA isolated from the saliva of the H. pylori infected subjects irrespective of their clinical status. The 16S rRNA amplification was carried out in the initial stages, along with the biopsy DNA, to confirm the presence of H. pylori in the saliva specimens of infected patients because 16S rRNA is the most conserved region to depict the presence of any organism 22 and also because of higher sensitivity and specificity of 16S rRNA primers to detect H. pylori in comparison with other primers specific to H. pylori. 14,23 Hence, in the present study, 16S rRNA amplification and histopathology were taken as a 'standard' to confirm active H. pylori infection.…”

Helicobacter pylori

“…Genes probed include those encoding urease: ureA; ureB and ureC [20]; and 16s ribosomal RNA. The latter can give a positive result with other Helicobacter species [30]. As a diagnostic tool PCR has two main advantages.…”

Loop‐Mediated Isothermal Amplification as a Fast Noninvasive Method of Helicobacter pylori Diagnosis

“…Because of the high clinical importance of H. pylori infection, numerous diagnostic methods based on nucleic acid technology for its detection in clinical, biological, and environmental samples have been developed (22,(26)(27)(28)(29)(30)(31)(32)(33)(34).…”