Next-Generation Nucleic Acid-Based Diagnostics for Viral Pathogens: Lessons Learned from the SARS-CoV-2 Pandemic
Abstract
1. Introduction
2. Coronavirus Epidemiology
3. Advances in Viral Nucleic Acid Detection Methods
3.1. Necessity–the Mother of Invention
3.2. Isothermal Amplification Methods (IAM)
3.2.1. Loop-Mediated Isothermal Amplification (LAMP)
3.2.2. Recombinase Polymerase Amplification (RPA)
3.2.3. Nicking Endonuclease Amplification Reaction (NEAR)
3.2.4. Rolling Circle Amplification (RCA)
3.2.5. Nucleic Acid Sequence-Based Amplification (NASBA)
3.3. Target Sensing Methods (TSM)
3.3.1. Specific High Sensitivity Enzymatic Reporter Unlocking (SHERLOCK)
3.3.2. DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) and All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR)
3.3.3. FnCAS9 Editor Linked Uniform Detection Assay (FELUDA)
3.3.4. Aptamer-Based Biosensors (Aptasensors)
4. Discussion
4.1. Complexities of Implementation
4.2. Cost of Implementation
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AOID-CRISPR | All-in-One-CRISPR |
CoV | Coronavirus |
COVID-19 | Coronavirus Infectious Disease-2019 |
CRISPR | Clustered Regularly Interspaced Short Palindromic Repeats |
DETTECTR | DNA Endonuclease Targeted CRISPR Trans Reporter |
EIA | Enzymatic ImmunoAssay |
FELUDA | FnCAS9 Editor Linked Uniform Detection Assay |
IAM | Isothermal Amplification Methods |
LAMP | Loop-Mediated Isothermal Amplification |
LFIA | Lateral Flow Immunoassay |
MERS | Middle East Respiratory Syndrome |
MRI | Magnetic Resonance Imaging |
NA | Nucleic Acid |
NASBA | Nucleic Acid Sequence-Based Amplification |
NEAR | Nicking Endonuclease Amplification Reaction |
PET/SPECT | Positron Emission Tomography/Single Photon Emission Computed Tomography |
POC | Point-of-Care |
RCA | Rolling Circle Amplification |
RNA | Ribonucleic Acid |
RPA | Recombinase Polymerase Amplification |
RTPCR | Real-Time reverse transcriptase Polymerase Chain Reaction |
SARS | Severe Acute Respiratory Syndrome |
SHERLOCK | Specific High Sensitivity Enzymatic Reporter Unlocking |
TSM | Target Sensing Methods |
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Method. | Template | Key Enzymes | Primer(s) | Temp. (°C) | Main Features |
---|---|---|---|---|---|
LAMP | DNA or RNA | Bst DNA polymerase (± RT) | 4–6 | 60–65 | High specificity, loop structures, turbidimetric/colorimetric detection |
RPA | DNA | Recombinase, SSB, strand-displacing DNA polymerase | 2 | 37–42 | Rapid (20–40min), low temperature, portable |
NEAR | DNA | strand-displacing DNA polymerase, nicking endonuclease | 2 | 55–59 | Ultra-rapid, uses endonuclease for nicking, suitable for point-of-care |
RCA | Circular DNA | Ligase, Phi29 DNA polymerase | 1 | 30–42 | Long concatemer products, simple design, high yield |
NASBA | RNA | Reverse transcriptase, T7 RNA polymerase, RNase H | 2 | 40–41 | RNA as the main target, transcription-based, isothermal |
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Papaneri, A.; Cui, G.; Chen, S.-H. Next-Generation Nucleic Acid-Based Diagnostics for Viral Pathogens: Lessons Learned from the SARS-CoV-2 Pandemic. Microorganisms 2025, 13, 1905. https://doi.org/10.3390/microorganisms13081905
Papaneri A, Cui G, Chen S-H. Next-Generation Nucleic Acid-Based Diagnostics for Viral Pathogens: Lessons Learned from the SARS-CoV-2 Pandemic. Microorganisms. 2025; 13(8):1905. https://doi.org/10.3390/microorganisms13081905
Chicago/Turabian StylePapaneri, Amy, Guohong Cui, and Shih-Heng Chen. 2025. "Next-Generation Nucleic Acid-Based Diagnostics for Viral Pathogens: Lessons Learned from the SARS-CoV-2 Pandemic" Microorganisms 13, no. 8: 1905. https://doi.org/10.3390/microorganisms13081905
APA StylePapaneri, A., Cui, G., & Chen, S.-H. (2025). Next-Generation Nucleic Acid-Based Diagnostics for Viral Pathogens: Lessons Learned from the SARS-CoV-2 Pandemic. Microorganisms, 13(8), 1905. https://doi.org/10.3390/microorganisms13081905