Sensitive fluorescence detection of pathogens based on target nucleic acid sequence-triggered transcription.

Accurate identification of mutant pathogens derived from genetic polymorphisms is highly desired in clinical diagnosis. However, current detection methods based on Watson-Crick hybridization suffers from false positives due to the cross-reactivity of wild-type sequences. In this study, we developed an accurate identification of mutant pathogens by combining programmable DNAzyme and target nucleic acid sequence-triggered transcription. Single nucleotide variants (SNVs) are the most plentiful type of mutations in the genome. High specificity to discriminate SNV was first achieved by rational design of dual-hairpin DNA structure and DNAzyme's capability of site-specific cleavage. T7 RNA polymerase-mediated transcription amplification was introduced to exponentially increase the sensitivity by encompassing T7 promoter sequence into the dual-hairpin DNA structure. The design of this biosensor is fast and straightforward without many computational steps, and the highly sensitive biosensor can detect not only SNVs but also occasional insertions and large deletions in the genome. We showed that the assay could rapidly detect COVID-19 variant and methicillin-resistant Staphylococcus aureus (MRSA), and the limit of detection is 0.96 copy/μL. The modular design of functional DNA enables this biosensor be easily reconfigured and is useful diagnosis of emerging infectious diseases caused by mutant pathogens.