Multiplexed Thermus thermophilus Argonaute-triggered tri-color fluorescent palette biosensing for rapid detection and genotyping of Helicobacter pylori

Helicobacter pylori ( H. pylori ) infection poses a significant threat to public health. In this work, we combined Thermus thermophilus A rgonaute ( Tt Ago) with l oop-mediated isothermal amplification (LAMP) to establish a t ri-color fluorescent biosensing for detection and genotyping of H . p ylori from human salivary samples, termed as ALT- Hp . Leveraging the programmability, site specific cleaving-activity of Tt Ago, ALT- Hp enabled the multiplexed detection of H. pylori -specific genes ureA , vacA , and cagA . Furthermore, a customized 3D-printed visualizer compatible with App-enabled smartphones was developed, highlighting its potential for on-site detection. ALT- Hp utilized the amplification capability of LAMP and the signal transduction and amplification “lighting-up” functions of Tt Ago, for building a highly sensitive, specific and robust platform for on-site detection. It demonstrated robust performance in complex matrices, achieving a LOD of 1 CFU/mL within 30 min. Clinical validation using 72 samples showed 100% sensitivity and specificity. ALT- Hp was not only able to detect H. pylori , but also capable of genotyping by classifying H. pylori as type I and type II. The classification is primarily based on whether the strains carry virulence factors, and the two types differed significantly in pathogenicity and clinical outcomes, serving as a critical reference for clinical diagnosis and treatment decision-making. Thus, this ALT- Hp biosensing represented a powerful molecular diagnostic tool for monitoring and management of H. pylori infections.