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A high-specificity flap probe-based isothermal nucleic acid amplification method based on recombinant FEN1-Bst DNA
1Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, PR China.
Biosensors & Bioelectronics
|July 24, 2021
Summary
A novel flap probe-based isothermal nucleic acid amplification method offers rapid, accurate detection of pathogens like SARS-CoV-2. This sensitive diagnostic tool aids in early disease identification and epidemic control.
Area of Science:
- Molecular Biology
- Biotechnology
- Infectious Disease Diagnostics
Background:
- The COVID-19 pandemic highlighted the need for rapid, accurate infectious disease diagnostics.
- Existing nucleic acid-based detection methods require improvement for early and effective epidemic control.
Purpose of the Study:
- To develop a novel, simplified, and highly specific isothermal nucleic acid amplification method.
- To evaluate the method's efficacy in detecting clinically relevant pathogens, including SARS-CoV-2.
Main Methods:
- Development of a flap probe-based isothermal amplification assay.
- Utilized engineered FEN1-Bst DNA polymerase with DNA synthesis, strand displacement, and cleavage functions.
- Tested detection of SARS-CoV-2 (ORF1ab and N genes), rotavirus, and Chlamydia trachomatis.
Main Results:
- Achieved limits of detection as low as 10 copies/μL for rotavirus, C. trachomatis, and SARS-CoV-2 N gene.
- Demonstrated 100% sensitivity and specificity compared to RT-PCR, with no cross-reactivity against 11 other pathogens.
- Enabled real-time detection and convenient endpoint display for point-of-care settings.
Conclusions:
- The novel nucleic acid sensor provides a simple, specific, and sensitive platform for pathogen detection.
- This method holds significant potential for clinical diagnostics, epidemic prevention, and control.
- The assay's adaptability for point-of-care use can improve infectious disease management.
