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

Area of Science:

• Biotechnology
• Molecular Biology
• Diagnostic Assays

Background:

• Accurate identification of mutant pathogens is crucial for clinical diagnosis.
• Existing hybridization methods yield false positives due to wild-type sequence cross-reactivity.
• Single nucleotide variants (SNVs) are common mutations requiring specific detection.

Purpose of the Study:

• To develop a highly specific and sensitive biosensor for identifying mutant pathogens.
• To overcome limitations of current diagnostic methods, particularly false positives.
• To enable rapid detection of various genetic mutations, including SNVs, insertions, and deletions.

Main Methods:

• Developed a biosensor combining programmable DNAzyme and target nucleic acid sequence-triggered transcription.
• Engineered a dual-hairpin DNA structure with DNAzyme for site-specific cleavage, ensuring SNV discrimination.
• Integrated T7 RNA polymerase-mediated transcription amplification for enhanced sensitivity.

Main Results:

• Achieved high specificity in discriminating SNVs through rational DNA structure design and DNAzyme cleavage.
• Demonstrated exponential sensitivity increase via transcription amplification.
• Successfully detected COVID-19 variants and methicillin-resistant Staphylococcus aureus (MRSA) with a limit of detection of 0.96 copy/μL.
• Showcased the biosensor's capability to detect insertions and large deletions.

Conclusions:

• The developed biosensor offers accurate and sensitive identification of mutant pathogens.
• Its modular design allows easy reconfiguration for diagnosing emerging infectious diseases.
• The assay is fast, straightforward, and applicable to diverse genetic variations in pathogens.