A one-pot RPA-T7/crDNA-CRISPR/Cas13a assay for portable and ultrasensitive detection of Helicobacter pylori and clarithromycin resistance mutations.

The high global prevalence of Helicobacter pylori (H. pylori) infection and rising clarithromycin resistance demand rapid, sensitive, point-of-care diagnostic tools. Although CRISPR/Cas systems have revolutionized nucleic acid detection, challenges such as unstable pre-synthesized crRNA and multi-step procedures limit their practical application. Here, we report a one-pot assay, termed RT-CRISPR (RPA-T7/crDNA-mediated CRISPR), that integrates recombinase polymerase amplification (RPA) with T7 transcription and CRISPR/Cas13a detection in a single closed tube. A key innovation is the use of a T7 promoter primers-crDNA hybrid chain (T7/crDNA) as a template for in situ synthesis of crRNA by T7 RNA polymerase, eliminating exogenous labile crRNA. The assay detects the H. pylori 16S rRNA gene at 1 copy/μL and the A2143G clarithromycin resistance mutation at 5 copies/μL, with no cross-reactivity against seven other gastrointestinal pathogens. It reliably detects the A2143G mutation at a ratio as low as 0.5% in mixed DNA (total 1× 104 copies/μL), highlighting its ability to identify low-abundance resistance mutations in heterogeneous samples. The entire assay, from DNA extraction to result, completes within 40 min using a portable fluorescence detector. Clinical validation with 103 samples showed 100% concordance with qPCR. The reagents can be lyophilized into stable pellets, maintaining performance after two months at room temperature. RT-CRISPR offers a simple, rapid, and robust platform for molecular diagnosis of H. pylori and drug resistance, holding promise for point-of-care testing and large-scale screening.