Harnessing recombinase polymerase amplification for rapid multi-gene detection of SARS-CoV-2 in resource-limited settings
| Autor: | Valérian Turbé, Dounia Cherkaoui, Rachel A. McKendry, Benjamin S. Miller, Da Huang |
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| Rok vydání: | 2021 |
| Předmět: |
Computer science
Biomedical Engineering Biophysics Loop-mediated isothermal amplification Recombinase Polymerase Amplification 02 engineering and technology Computational biology Biosensing Techniques Real-Time Polymerase Chain Reaction 01 natural sciences Sensitivity and Specificity Recombinases Electrochemistry Humans Gene Pandemics SARS-CoV-2 010401 analytical chemistry COVID-19 General Medicine Dipstick Gold standard (test) Nucleic acid amplification technique 021001 nanoscience & nanotechnology Reverse transcriptase 0104 chemical sciences Real-time polymerase chain reaction Molecular Diagnostic Techniques RNA Viral 0210 nano-technology Nucleic Acid Amplification Techniques Biotechnology |
| Zdroj: | Biosensorsbioelectronics. 189 |
| ISSN: | 1873-4235 |
| Popis: | The COVID-19 pandemic is challenging diagnostic testing capacity worldwide. The mass testing needed to limit the spread of the virus requires new molecular diagnostic tests to dramatically widen access at the point-of-care in resource-limited settings. Isothermal molecular assays have emerged as a promising technology, given the faster turn-around time and minimal equipment compared to gold standard laboratory PCR methods. However, unlike PCR, they do not typically target multiple SARS-CoV-2 genes, risking sensitivity and specificity. Moreover, they often require multiple steps thus adding complexity and delays. Here we develop a multiplexed, 1-2 step, fast (20-30 min) SARS-CoV-2 molecular test using reverse transcription recombinase polymerase amplification to simultaneously detect two conserved targets - the E and RdRP genes. The agile multi-gene platform offers two complementary detection methods: real-time fluorescence or dipstick. The analytical sensitivity of the fluorescence test was 9.5 (95% CI: 7.0-18) RNA copies per reaction for the E gene and 17 (95% CI: 11-93) RNA copies per reaction for the RdRP gene. The analytical sensitivity for the dipstick method was 130 (95% CI: 82-500) RNA copies per reaction. High specificity was found against common seasonal coronaviruses, SARS-CoV and MERS-CoV model samples. The dipstick readout demonstrated potential for point-of-care testing in decentralised settings, with minimal or equipment-free incubation methods and a user-friendly prototype smartphone application. This rapid, simple, ultrasensitive and multiplexed molecular test offers valuable advantages over gold standard tests and in future could be configurated to detect emerging variants of concern. |
| Databáze: | OpenAIRE |
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