Quantitative polymerase chain reaction for the detection of Helicobacter pylori in gastric biopsy specimens

Abstract

A variety of methods, including the polymerase chain reaction (PCR), are available for the detection of Helicobacter pylori in clinical samples, but none of them can adequately quantify the organism. In the present study, the competitive PCR, a rapid and simple method for quantification of Helicobacter pylori DNA in gastric biopsies, was used to measure the amount of DNA present in Helicobacter pylori-positive biopsies. This method is based on coamplification of an internal standard and a target DNA sequence with one set of primers. The internal standard was prepared using a nonhomologous fragment of DNA ligated to specific primers used to amplify the target DNA. This competitive DNA fragment of a desired size and containing primer templates is called a PCR MIMIC. To perform a quantitative PCR, PCR amplification reactions were spiked with known quantities of PCR MIMICs containing unknown amounts of DNA from Helicobacter pylori-positive biopsies. The amount of target DNA was determined by visual comparison of the PCR products after establishment of the correlation between the internal control concentration and the DNA concentration in a competitive amplification reaction. The results were confirmed by a radioactive method. Quantitative PCR can be a reliable method for determining the extent of Helicobacter pylori infection.