Microfluidic nano-scale qPCR enables ultra-sensitive detection of SARS-CoV-2

Abstract

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.

Nanotechnology and COVID-19: Prevention, diagnosis, vaccine, and treatment strategies

“…The major reason why inorganic NPs need modification is concerns about toxicity. Due to the close morphological and physicochemical properties of SARS-CoV-2 and synthetic NPs, it allows NPs to be an effective intervention method (Wang J. et al, 2020;Xie et al, 2020;Behbudi, 2021;Doagooyan, 2021;El-Megharbel et al, 2021).…”

Biomedical Applications of Microfluidic Devices: A Review

“…Currently, there are three main microfluidics biosensors developed to detect nucleic acid: PCR, (CRISPR)/clustered regularly interspaced short palindromic repeats, and isothermal amplification [ 465 ]. The microfluidics-integrated qPCR method that was reported by several research groups has the advantage of high throughput scheme processing, allowing massive quantitative analysis, including preparation and detection in a single chip platform [ 466 , 467 ]. A recent PCR-based microfluidic system introduced by Cojocaru et al was a disposable chip functionalized with lyophilized probes and primers, which offered a rapid result (less than 30 mins) for as much as 1.2 μL volume per reaction [ 468 ].…”