Microfluidic Nano-Scale qPCR Enables Ultra-Sensitive and Quantitative Detection of SARS-CoV-2
| Autor: | Mohammed Zafar, Fabio Piano, Yasmine Moussa, Xin Xie, Kristin C. Gunsalus, Zyrone Victoria, Mame Massar Dieng, Khristine Pamplona, Zaynoun Attieh, Christopher A. Jackson, Youssef Idaghdour, Mostafa Khair, Marc Arnoux, Raghib Ali, Fatima Al Jallaf, Lobna El Messery, Nabil Rahiman, Tamara Gjorgjieva |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Coronavirus disease 2019 (COVID-19) viruses Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 030106 microbiology Microfluidics microfluidics detection Bioengineering lcsh:Chemical technology nano-qPCR lcsh:Chemistry 03 medical and health sciences 0302 clinical medicine Chemical Engineering (miscellaneous) lcsh:TP1-1185 030212 general & internal medicine ultra-sensitive Ultra sensitive Detection limit Chromatography Chemistry SARS-CoV-2 Process Chemistry and Technology COVID-19 Orders of magnitude (mass) viral RNA viral load lcsh:QD1-999 Clinical diagnosis Viral load |
| Zdroj: | Processes Volume 8 Issue 11 Processes, Vol 8, Iss 1425, p 1425 (2020) |
| ISSN: | 2227-9717 |
| DOI: | 10.3390/pr8111425 |
| Popis: | A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. In this study, we implemented a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification, and nano-scale qPCR based on a commercially available microfluidic chip. Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of this microfluidic RT-qPCR by 1000-fold, enabling detection below 1 copy/µ L. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible and quantitative detection of SARS-CoV-2 over five orders of magnitude (< 1 to 106 viral copies/µ L). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (< 1 to 40 viral copies/µ L) in 17 samples with negative clinical diagnosis, indicating a potential false-negative rate of 18.7% by clinical diagnostic procedures. In summary, this three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (< 1 viral copy/µ L) and has the potential to reduce the false-negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections. |
| Databáze: | OpenAIRE |
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