Synonymous codon-optimized multiplex qPCR for simultaneous detection of <i>Helicobacter pylori</i> and precise drug resistance profiling in infected individuals

ABSTRACT Antibiotic-resistant Helicobacter pylori ( H. pylori ) represents a significant global challenge to the efficacy of eradication therapies, underscoring the urgent need for rapid and precise pretreatment resistance profiling. Traditional genotyping assays frequently neglect synonymous mutations, resulting in false-negative results and reduced diagnostic accuracy. In this study, we developed a single-tube, synonymous codon-optimized multiplex real-time PCR (qPCR) assay for the qualitative detection of H. pylori and the simultaneous identification of mutations associated with resistance to clarithromycin (23S rRNA gene) and levofloxacin ( gyrA gene). The probe design was informed by clinical mutation data and synonymous codon usage patterns from 453 isolates, achieving over 98% coverage of naturally occurring variants. The assay exhibited high sensitivity (limit of detection: 2–20 copies/μL), excellent repeatability (coefficient of variation &lt;5%), and robust specificity. Clinical validation using 316 gastric mucosal specimens demonstrated 97.47% concordance with a commercial infection detection kit and 100% and 98.08% agreement with Sanger sequencing for 23S rRNA and gyrA resistance genotyping, respectively. This assay effectively addresses the critical limitation posed by synonymous mutations, providing a rapid, reliable, and clinically accessible tool to guide personalized H. pylori eradication therapy and promote antimicrobial stewardship. IMPORTANCE Increasing antibiotic resistance in Helicobacter pylori has reduced the effectiveness of eradication therapy and highlights the need for accurate resistance profiling before treatment. However, current molecular assays may miss resistant strains because synonymous mutations in probe-binding regions may interfere with detection and produce false-negative results. In this study, we developed a single-tube multiplex real-time PCR assay that incorporates synonymous codon variation into probe design, enabling simultaneous detection of H. pylori infection and clarithromycin- and levofloxacin-resistance mutations. By using sequence data from 453 clinical isolates, this assay achieved broad coverage of naturally occurring variants and showed high analytical and clinical performance. This approach addresses an important diagnostic blind spot in H. pylori resistance detection and provides a practical tool for pretreatment resistance profiling, individualized therapy selection, and improved antimicrobial stewardship.