Development of a real-time polymerase chain reaction (RT-PCR) assay for simultaneous detection of Helicobacter pylori infection and genetic mutations associated with clarithromycin resistance: A single-center, cross-sectional study from Singapore

Abstract

Purpose

Accurate diagnosis of Helicobacter pylori (H. pylori), including detection of clarithromycin (CAM) resistance, is important for successful disease management. This study aimed to develop a real-time PCR assay that can simultaneously detect H. pylori infection and screen for point mutations in the 23S rRNA gene responsible for CAM resistance.

Methodology

A probe-based assay based on fluorescence melting curve analysis (FMCA) was developed and evaluated in this study. Primers flanking a 173 bp region of the 23S rRNA gene were used for the target amplification. The probe was designed to cross over the mutation hotspots, e.g. A2142G, A2143G, to detect point mutations in the melting stage.

Results

A total of 410 gastric tissue biopsies were cultured for H. pylori and concurrently tested by the PCR assay. Among these samples, 407 samples had a valid PCR result. 270 confirmed samples were H. pylori-positive by both PCR and culture. 113 samples were H. pylori-negative by both PCR and culture, and 24 samples were H. pylori-positive by PCR but negative by culture. For susceptibility testing, genotypic results were compared with phenotypic results. In the phenotypic CAM-susceptible group (n = 197), PCR FMCA correctly categorised 95.4 % (n = 188) of the samples into CAM-susceptible genotype. In the phenotypic CAM-resistant group (n = 64), PCR FMCA correctly classified 56 samples as CAM-resistant genotype, equal to 87.5 % accuracy.

Conclusions

This PCR assay provides an accurate method to detect H. pylori infection and simultaneously detect mutations in the 23S rRNA gene that are associated with CAM resistance.