Abstract

One-tube RPA-PfAgo assay for simultaneous Hp detection and VacA genotyping. • LOD of 10 copies/μL for UreA and VacA, 10-fold more sensitive than qPCR. • Clinical validation on saliva samples showed 94.1% sensitivity and 100% specificity. • Non-invasive detection completed within 50 min with minimal equipment requirement. Gastric carcinoma (GC) remains a leading cause of global cancer-related mortality, with Helicobacter pylori (Hp) infection identified as its primary risk factor. It is classified as a Group I carcinogen by the World Health Organization. Current Hp detection methods are limited by poor genotyping precision, high false-positive rates, and reliance on invasive sampling, which hinder early risk stratification. Here, we developed a molecular assay that integrates Recombinase Polymerase Amplification (RPA) and Pyrococcus furiosus Argonaute ( Pf Ago) for specific detection of the Hp virulence factor VacA. We designed RPA primers targeting conserved sequences of the ureA and vacA genes, followed by guide DNA (gDNA) and fluorescent probes. The one-tube RPA- Pf Ago system was optimized using Hp genomic DNA, and its performance was validated with clinical saliva samples. The assay demonstrated a LOD of 10 copies/μL for both ureA and vacA , demonstrating a 10-fold higher sensitivity than quantitative PCR (qPCR, LOD = 10 2 copies/μL). Clinical validation (n = 26 identified saliva samples) showed 94.1% sensitivity and 100% specificity, enabling accurate differentiation of VacA-positive high-virulence strains. This method not only avoids invasive sampling but also reduces detection time to 50 min. The RPA- Pf Ago assay provides a rapid, non-invasive tool for Hp screening and virulence typing, facilitating personalized GC risk assessment and clinical management. The established technical framework shows good scalability for other pathogen detection, public health surveillance, and food safety monitoring scenarios. Download: Download high-res image (3