Evaluation of a Novel Heminested PCR Assay Based on the Phosphoglucosamine Mutase Gene for Detection of Helicobacter pylori in Saliva and Dental Plaque

Abstract

ABSTRACT A novel heminested PCR protocol was developed for the specific detection of Helicobacter pylori at low copy numbers. A set of primers specific for the phosphoglucosamine mutase gene ( glmM ) of H. pylori produced a 765-bp fragment that was used as template for the heminested primer pair delineating a 496-bp fragment. By using agarose gel electrophoresis for detection of the heminested PCR-amplified products, amplification of H. pylori genomic DNA was achieved at concentrations as low as 0.1 pg, equivalent to 5 × 10 2 bacteria. A study was subsequently undertaken to evaluate the heminested PCR for detection of H. pylori in dental plaque and saliva. Specimens collected from 58 individuals were cultured, and PCR was subsequently performed on the oral cultures. Identification of H. pylori in the same series of saliva and dental plaque specimens was carried out with PCR using a primer pair specific for the H. pylori urease B gene and by the heminested PCR assay. The identity of the amplified products was confirmed by DNA sequencing. Our results demonstrate that the heminested PCR assay was specific for detection of H. pylori , yielding no false-positive results, and that H. pylori had a low prevalence (approximately 3%) in specimens obtained from the oral cavity.