Asy-RPA/PCR combined with One-crRNA-CRISPR/Cas12a for simultaneous detection of multiple Clarithromycin resistance mutations in Helicobacter pylori.

METHODS

Genetic testing for Clarithromycin resistance-associated single-nucleotide variations (SNVs) in H. pylori could be applied for formulating individual eradication plan. In this study, we integrated asymmetric recombinase polymerase amplification (Asy-RPA) with a single crRNA for CRISPR/Cas12a-designated the ARoRC system-to circumvent protospacer adjacent motif (PAM) dependency.

RESULTS

The ARoRC platform detected all targeted mutations with 100% agreement compared to Sanger sequencing. Assay sensitivity was determined as follows: A2143G (10-2 ng/μL), A2142C (2.58 × 10-3 ng/μL), A2142G (2.49 × 10-3 ng/μL), and A2142G + A2143G (2.39 × 10-3 ng/μL), enabling Asy-PCR-CRISPR/Cas12a detection suitable for fecal samples. The assay achieved visual results within 1 h using lateral flow strips, with no cross-reactivity to WT or non-target sequences.

DISCUSSION: We developed a rapid, ultrasensitive, and portable assay for detecting Clarithromycin resistance-associated mutations in H. pylori. The robustness of the platform in complex matrices such as feces, along with its dual readout capability (fluorescence and lateral flow), supports its potential for point-of-care (POC) application.