Quantitation of Helicobacter pylori in the Stomach Using Quantitative Polymerase Chain Reaction Assays

Abstract

The ability to rapidly quantitate H. pylori from gastric mucosa should be useful to investigate the role of H. pylori density and infection outcome, as well as to monitor the effectiveness of antibiotic treatment or vaccines against H. pylori.

Real-Time Quantitative PCR for Detection ofHelicobacter pylori

“…To isolate H. pylori DNA from fresh or frozen biopsy samples of the stomach, three methods have been used: kits from commercial companies, the proteinase K-phenol method, and a boiling-water method. The best of the three methods for obtaining a high yield or purity of H. pylori DNA has been shown to be the classical proteinase K-phenol method (10,18,19,25,44,51,56), which is supported by our results. Although we showed little difference in DNA degradation between DNA isolated with the commercial kit and that isolated with the revised proteinase K-phenol method, the revised proteinase K-phenol method produced a much higher yield.…”

“…Although the kinetic methods have several advantages over the coamplification methods (13,43), the few published studies of quantitation of H. pylori in biopsy samples (18,39,44,45) have used the coamplification method. This is the first report of real-time Q-PCR to detect H. pylori.…”

“…The specificity of Q-PCR amplification of this fragment of the ureC gene has been tested previously in our laboratory (44). No PCR occurred with the ureC primers with DNA extracts from normal mouse stomachs (not infected with H. pylori), DNA extracts from normal mouse stomach flora grown on Luria-Bertani (LB) agar plates (44), or human genomic placental DNA.…”

“…The specificity of Q-PCR amplification of this fragment of the ureC gene has been tested previously in our laboratory (44). No PCR occurred with the ureC primers with DNA extracts from normal mouse stomachs (not infected with H. pylori), DNA extracts from normal mouse stomach flora grown on Luria-Bertani (LB) agar plates (44), or human genomic placental DNA. Additionally, the sequencing results from two human stomach biopsy samples matched the H. pylori ureC gene but no genes from any other organism, as shown by Blast analysis on the National Center for Biotechnology Information web site.…”

“…The PCR products can be measured by the intensity of ethidium bromide luminescence on agarose gels (18), an enzyme-linked immunosorbent assay (ELISA) after capturing biotin-labeled probe-amplicon complexes on streptavidin-coated microtiter plates, or an ELISA-based bioluminescence assay in which a calcium-dependent flash-type bioluminescent tag (aequorin) is used to detect the product at the attomolar level (1, 2). The last two detection techniques have been used in our laboratory (44,46). Furthermore, coamplification methods involving competitive and noncompetitive PCR use endpoint analysis, which is adversely affected by variable amplification efficiency (13,43).…”