One-Pot LAMP-Coupled CRISPR/Cas12b Assay Enables Sensitive Detection of Helicobacter 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 the CagA gene was developed for H. pylori detection. 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 × 101 copies/µL, representing a tenfold improvement in sensitivity compared with conventional LAMP and PCR assays (3.14 × 102 copies/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 specificity, rapid turnaround, and operational simplicity, demonstrating strong potential for point-of-care testing and applications in resource-limited settings.