Identification of multiple single-nucleotide variants for clinical evaluation of Helicobacter pylori drug resistance.

Helicobacter pylori infections are threats to public health due to their high infection rate and drug resistance. Identification of single-nucleotide variants (SNVs) in H. pylori is crucial for both diagnosis and therapy. Yet the clinical testing of resistant H. pylori mutants is still facing some challenges, such as the selectivity is not good enough for SNVs in abundant wild-type DNA, the lack of clinical validation and the economical burden on patients. Herein, an X-shaped DNA probe with a toehold initiator was designed, which could specifically hybridize with certain genotype DNA due to the thermodynamically driven reaction. A competitive reaction was developed to amplify the thermodynamic difference between wild-type DNA and SNVs, diminishing the interference of wild-type DNA. By this means, multiple SNVs in H. pylori were successfully identified and two SNVs related to clarithromycin resistance are chosen as model targets. A paper strip was fabricated for visual, fast screening of SNVs. Furthermore, the approach was validated using clinical samples, and a point-of-care (POCT) testing diagnosis was executed on saliva samples, demonstrating its potential for the prevention and cure of H. pylori infections.