Abstract

Simple SummaryEarly and efficient detection ofHelicobacter pyloriis critical for gastric cancer prevention, particularly in resource-limited settings. Current diagnostic methods, however, remain inadequate for rapid, on-site testing. Here, we developed a one-pot LAMP-CRISPR/Cas12b system targeting the conservedCagAgene, combining rapid isothermal amplification with Cas12b-mediated signal amplification. The assay can be completed within one hour, achieves a tenfold higher sensitivity compared to quantitative polymerase chain reaction or loop-mediated isothermal amplification alone, and shows no cross-reactivity with other pathogens. This method provides a powerful tool for point-of-care testing and clinical diagnosis ofHelicobacter pylori.Helicobacter pylori(H. pylori) infection is closely associated with the development of chronic gastritis, peptic ulcers, and gastric cancer, highlighting the importance of rapid and accurate detection for disease prevention and clinical management. In this study, a one-pot LAMP-CRISPR/Cas12b assay targeting theCagAgene was developed forH. pyloridetection. First, the LAMP system was optimized by systematically screening key reaction components. Subsequently, a one-step LAMP-CRISPR/Cas12b detection platform was established through optimization of the ratio between the LAMP premix and CRISPR buffer, reaction temperature, Cas12b concentration, and ssDNA reporter concentration. Under optimal conditions, the assay achieved a detection limit of 3.14 × 101copies/µL, representing a tenfold improvement in sensitivity compared with conventional LAMP and PCR assays (3.14 × 102copies/µL). In addition, the entire detection process could be completed within 1 h. Validation using 17 culture-positive and 17 culture-negative samples demonstrated complete concordance with culture-based results, with no false-positive or false-negative detections observed. These findings indicate that the proposed platform possesses high sensitivity, excellent specifi