A rapid and high-throughput Helicobacter pylori RPA-CRISPR/Cas12a-based nucleic acid detection system

Abstract

BackgroundHelicobacter pylori lives in the human stomach and causes gastric cancer and other gastric diseases. The development of molecular technology has facilitated low-cost, rapid, and high-throughput detection of H. pylori. Materials and methodsThe combination of isothermal recombinase polymerase amplification (RPA) and CRISPR-Cas12a was used for early diagnosis and monitoring of H. pylori in clinical settings. The UreB genes from 242 H. pylori strains were subjected to cluster analysis, and we designed corresponding RPA primers and screened 2 sets of CRISPR-derived RNAs (crRNAs) for accurate H. pylori recognition. We then performed specificity and sensitivity validation of seven strains using this RPA-CRISPR/Cas12a method. In addition, the cut-off values of this RPA-CRISPR/Cas12a method based on fluorescence values (i.e., RPA-CRISPR/Cas12a-FT) were determined by comparison with quantitative PCR (qPCR), and further experiments comparing different methods were performed using clinical samples. ResultsWe developed a rapid detection system based on the combination of RPA and CRISPR-Cas12a, which was applied to the early diagnosis and monitoring of H. pylori in clinical settings. The RPA-CRISPR/Cas12a system was used to detect the UreB gene. We found that the limit of detection (LOD) for the CRISPR/Cas12a method based on the lateral flow dipstick result (i.e., CRISPR/Cas12a-LFD) was 100 copies, the cut-off value was 1.4; and for CRISPR/Cas12a-FT the LOD was 50 copies. This system was used to assess clinical samples and showed high reproducibility with proof-of-concept sensitivity, and the whole detection process was completed within 40 min. ConclusionAs a diagnostic method that can detect the UreB gene of H. pylori in gastric tissue samples rapidly, sensitively, visually, and in a high throughput manner, our method provides a new diagnostic option for clinicians. This system is ideal for hospitals or testing sites with limited medical resources.