Abstract

Purpose This study aims to establish a platform and to achieve rapid, simultaneous detection of Helicobacter pylori along with its key virulence genes, cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (vacA) . Methods We developed a multiplex recombinase polymerase amplification combined with lateral flow strip (mRPA-LFS) assay for the rapid, simultaneous detection of H. pylori (targeting the 16 S rRNA gene) and its key virulence genes, cagA and vacA . The assay employs a nucleic acid tag (NAT)-bridging strategy for multiplex visualization without instrumentation. Performance was evaluated using recombinant plasmids and clinical saliva samples ( n  = 28), with 16 S rRNA detection compared against the urea breath test and genotyping results against quantitative PCR (qPCR). Results The mRPA-LFS assay completes detection within 30 min at a constant temperature of 39 °C. It demonstrated high sensitivity with limits of detection of 10¹, 10³, and 10² copies/µL for the 16 S rRNA , cagA , and vacA genes, respectively, and showed no cross-reactivity with nine common non- H. pylori pathogens. The detection results of the mRPA-LFS for H. pylori were highly consistent with those of qPCR (complete agreement for 16 S rRNA and cagA genes; sensitivity of 94.4% and specificity of 100% for vacA ). Conclusions The established mRPA-LFS platform represents a significant advance toward point-of-care molecular typing of H. pylori , offering a simple, rapid, and multiplex strategy that is particularly suitable for frontline screening and clinical decision-making in low-resource environments.