A novel CRISPR/Cas12a biosensor for sensitive detection of Helicobacter pylori from clinical patients

Sensitive, simple, and inexpensive detection plays an important role in clinical practice. Herein, we constructed a novel CRISPR-Cas12a biosensor for the detection of Helicobacter pylori (H. pylori) from clinical patients. We selected and synthesized 16 S rRNA sequences of H. pylori to develop the CRISPR-Cas12a biosensor. A novel DNA isothermal amplification technique termed hairpin-mediated self-primer exponential amplification (HSEA) was employed to amplify the detection target. The hairpin probe recognized and hybridized with the target sequence and then formed a self-primer at the 3' end. The 3' terminus of the hairpin-target duplex would be extended by DNA polymerase under isothermal conditions to form an extended hairpin (EHP1). The target sequence would be displaced during the polymerization process to initiate another HSEA cycle. 5' terminus of EHP1 with phosphorothioate (PS) modification could form a hairpin structure due to the lower Tm value. Theoretically, the amplified hairpin can always maintain its own primer-forming and extension state to produce a long-extended hairpin containing numerous target sequence domains (EHPX). The amplification products were recognized by CRISPR/Cas12a to release the fluorescence signal. This HSEA-CRISPR/Cas12a biosensor allows to detect H. pylori strain ATCC 26695 16 S rRNA gene within 1.5 hours with a detection limit of 70 ng/μL. For clinical sample detection, this HSEA-CRISPR/Cas12a biosensor accurately identified H. pylori (22/26 agreement). This method shows high specificity in differentiating H. pylori from very closely related bacteria. The assay is rapid (sample-to-answer times less than 1.5 hours), cost-effective, and sensitive.