Quantitation ofHelicobacter pyloriin dental plaque samples by competitive polymerase chain reaction

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Abstract
Aim—To establish a competitive PCR (cPCR) assay for quantitation ofH pyloriorganisms in dental plaque samples.Methods—The cPCR coamplified targetH pyloriDNA and a known amount of internal standard template in the same tube with the same primers directed to 0.86 kb DNA of H pylori. The internal standard was a synthesised DNA bearing the same primer recognition sites at two ends and a non-homologous core sequence as the target DNA fragment. Quantitation was based on determination of the relative, not absolute, amounts of the differently sized and [32P]-dCTP labelled products derived fromH pyloriDNA and the competitive internal standard after gel electrophoresis separation.Results—A significant correlation between known amounts ofH pyloriadded to dental plaque samples and the results of the cPCR was found, and a standard line was developed which allowed quantitation ofH pyloriin the plaque samples. cPCR was performed on supragingival plaque samples from 10 adult patients with H pylori infection in the stomach, and from five adults and six children withoutH pyloriinfection in the stomach. The ranges ofH pylorinumbers were 1–213 (median 25), 6–76 (10), and 4–94 (14) cells/mg of dental plaque in the three groups, respectively.Conclusions—cPCR is useful for quantitation ofH pyloriin supragingival dental plaque samples; however, the number of the organisms in dental plaque samples seems very low.