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/µL. 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/µL). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/µL) 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/µL) 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.
Background: A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used standard 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. Methods: We implement a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification and nano-scale qPCR based on the Fluidigm 192.24 microfluidic chip. We validate the method using both positive controls and nasopharyngeal swab samples. Results: Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of the Fluidigm method by 1,000-fold, enabling detection below 1 copy/μl. 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 detection of SARS-CoV-2 over five orders of magnitude (< 1 to 106 viral copies/μl). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/μl) in 17 samples with negative clinical diagnosis, indicating a potential false negative rate of 18.7% by clinical diagnostic procedures. Conclusion: The three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (< 1 viral copy/μl) 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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.