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Sensitive fluorescent detection of SARS-CoV-2 RNA using an enzymatic-based method.
André Miranda1, Bruno Baptista1, Marília Figueira2
1CICS-UBI - Health Sciences Research Centre University of Beira Interior Covilhã Portugal; RISE-Health, Departamento de Química, Faculdade de Ciências, Universidade da Beira Interior, Rua Marquês d'Ávila e Bolama 6201-001 Covilhã, Portugal.
This study presents a rapid hairpin-mediated nicking enzymatic signal amplification (HNESA) method for detecting SARS-CoV-2 RNA. The technique offers a quick, sensitive, and complementary diagnostic approach for viral infections.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Accurate detection of viral oligonucleotides is crucial for timely diagnosis and management of infections.
- Existing methods like PCR can be time-consuming.
- A rapid, sensitive nucleic acid detection method is needed.
Purpose of the Study:
- To adapt the hairpin-mediated nicking enzymatic signal amplification (HNESA) method for SARS-CoV-2 RNA detection.
- To develop a rapid and sensitive diagnostic tool for viral diseases.
Main Methods:
- Designed an assistant probe (AP) with target complementarity, molecular beacon (MB) hybridization site, and nicking endonuclease recognition site.
- Optimized MB sequences (MB1 and MB2) for length and nucleotide composition.
- Utilized the HNESA method for cyclic amplification and fluorescence signal generation.
Main Results:
- The optimized HNESA method demonstrated significant fluorescence signal amplification with MB2.
- Achieved a linear detection range from 0.1 to 1 nM, with a detection limit of 170.6 pM.
- Obtained results within 45 minutes, highlighting the method's speed and efficiency.
Conclusions:
- The HNESA method is a rapid, efficient, and highly sensitive technique for SARS-CoV-2 RNA detection.
- This approach can serve as a valuable complementary diagnostic tool to polymerase chain reaction (PCR).
- The HNESA method shows potential for diagnosing various viral diseases.
