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

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.

A Population Genetics Pedigree Perspective on the Transmission of Helicobacter pylori

“…Sampling and gene sequencing: The study population comprised 105 healthy individuals, for which extensive pedigree information was available (supplemental Figure 1 at http:/ /www.genetics.org/supplemental/), from a rural, South African, black community (Ogies, Mpumulanga) who have been followed as part of a long-term surveillance program on the epidemiology of H. pylori (Goosen et al 2002;Fritz et al 2006;Olivier et al 2006). This population had many of the risk factors that are associated with a high prevalence of H. pylori infection (Bunn et al 2002;Suerbaum and Michetti 2002).…”

“…This infection is associated with chronic gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma, has a major impact on public health (Alm et al 2000), and has been classified as a group I carcinogen by the International Agency for Research on Cancer (NIH Consensus Development Panel 1994). Although much is known about the virulence of H. pylori (Covacci et al 1999), the potential transmission pathways for the bacterium are unresolved (Goosen et al 2002;Dowsett and Kowolik 2003). Potential transmission routes include oral-oral and fecal-oral, both with and without intermediate transmission steps.…”

Correlation of 16s rRNA with serum levels of the cytokines, TNF-α and IL-1β, in subjects with a positive Helicobacter pylori Stool Antigen test (HPSA)

“…The accuracy of diagnosis of H. pylori infection with the use of stool PCR varies from 25 to 100% due to stool inhibitors or organism decomposition in the intestines, but since this is a non-invasive method, it is preferable to invasive procedures (6). In addition to pathogenicity of the bacterium and its impact on the disease, recent studies have shown that host genetic and environmental factors are significantly involved in the incidence of H. pylori infection (7,8). For example, in a study by Oba-Shinjo and Zhang in 2004 and 2008, it was observed that the presence of cytokines such as TNF-α and IL-1β is important in predicting susceptibility to H. pylori infection (9).…”

A South African perspective on Helicobacter pylori: Prevalence, epidemiology and antimicrobial chemotherapy

“…The oral cavity was evaluated as a reservoir from where H. pylori may be transmitted however; the organism was not detected in dental samples . On the contrary, prevalence as low as approximately 3% was reported in specimens obtained from the oral cavity in another study in Pretoria (Goosen et al, 2002). Although a number of studies have demonstrated the role of gastro-oral, iatrogenic, sexual transmissions, horizontal transfer and intrafamilial clustering (Brown, 2000;Eslick, 2000;2002;Delport et al, 2006;Fritz et al, 2006;Kast, 2007) in the transmission of H. pylori; no studies have been done in South Africa to ascertain the role of these routes in the transmission of the organism.…”

“…Louw et al (2001) in their study in Cape Town documented an H. pylori prevalence that ranged from 60% using the rapid urease test to 79% with serology. A low prevalence (approximately 3%) of H. pylori was reported in specimens obtained from the oral cavity in a study by Goosen et al (2002) in Pretoria.…”