Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples

Abstract

The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the BiomarkTM instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of BiomarkTM reactions is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several protocols, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.

SARS-CoV-2 Diagnostics Based on Nucleic Acids Amplification: From Fundamental Concepts to Applications and Beyond

“…Microfluidics is based on the manipulation of fluids at a sub-millimeter scale, having multiple applications in molecular diagnostic with a growing number of solutions trusting on microfluidic-related platforms that contain nucleic acid amplification stages ( Anderson et al., 2021 ; Fassy et al., 2021 ; Liu et al., 2021 ; Xing et al., 2021 ). The higher cost and complexity of these systems, which are often used at the point-of-care ( Table 7 ), in comparison to LFA-based methods, are challenges that may discourage its use ( Liu et al., 2021 ).…”

“…Microfluidic systems can be further divided in various subsets, of what rotationally-driven ( Ji et al., 2020 ; Xiong et al., 2020 ; de Oliveira et al., 2021 ), electric field-driven ( Ramachandran et al., 2020 ) and paper-based ( Garneret et al., 2021 ) options were already adopted in the context of SARS-CoV-2. The normal workflow used for detecting viral RNA in other approaches, which includes extraction, amplification and detection of nucleic acids can be partially ( de Oliveira et al., 2021 ; Fassy et al., 2021 ; Liu et al., 2021 ), or entirely ( Garneret et al., 2021 ) conducted in microfluidic platforms, since the extraction step is often conducted out of the microfluics apparatus. There are different types of amplification strategies already assessed in these devices that rely on PCR-related methods, generally qRT-PCR ( Anderson et al., 2021 ; Fassy et al., 2021 ; Xing et al., 2021 ), or isothermal amplification techniques related to LAMP ( Ramachandran et al., 2020 ; Xiong et al., 2020 ; de Oliveira et al., 2021 ; Garneret et al., 2021 ), NASBA ( Xing et al., 2020 ), RPA ( Liu et al., 2021 ) and RCA ( Kim et al., 2021 ), including approaches based on CRISPR-Cas ( Ramachandran et al., 2020 ).…”

“…The normal workflow used for detecting viral RNA in other approaches, which includes extraction, amplification and detection of nucleic acids can be partially ( de Oliveira et al., 2021 ; Fassy et al., 2021 ; Liu et al., 2021 ), or entirely ( Garneret et al., 2021 ) conducted in microfluidic platforms, since the extraction step is often conducted out of the microfluics apparatus. There are different types of amplification strategies already assessed in these devices that rely on PCR-related methods, generally qRT-PCR ( Anderson et al., 2021 ; Fassy et al., 2021 ; Xing et al., 2021 ), or isothermal amplification techniques related to LAMP ( Ramachandran et al., 2020 ; Xiong et al., 2020 ; de Oliveira et al., 2021 ; Garneret et al., 2021 ), NASBA ( Xing et al., 2020 ), RPA ( Liu et al., 2021 ) and RCA ( Kim et al., 2021 ), including approaches based on CRISPR-Cas ( Ramachandran et al., 2020 ). In the context of SARS-CoV-2 diagnostics, the detection of DNA following the amplification reaction is usually performed by integrated measuring of fluorescence ( Ramachandran et al., 2020 ; Xing et al., 2020 ; de Oliveira et al., 2021 ; Fassy et al., 2021 ; Garneret et al., 2021 ), although LFAs ( Liu et al., 2021 ), or rheometry devices ( Kim et al., 2021 ) have already been combined with microfluidic systems for this same purpose.…”

Evaluation of the Advanta Dx SARS-CoV-2 RT-PCR Assay, a High-Throughput Extraction-Free Diagnostic Test for the Detection of SARS-CoV-2 in Saliva: A Diagnostic Accuracy Study

“…With regard to analytical sensitivity, the Limit of Detection (LoD) for the Advanta Dx SARS-CoV-2 RT-PCR Assay was estimated to be 6.25 genome equivalents (GE)/µL according to the instructions for use document [6].…”

Automated ELISA On-Chip for the Detection of Anti-SARS-CoV-2 Antibodies

“…Despite this, various microfluidic devices have been developed in response to the problems posed by the COVID-19 pandemic. For example, in the diagnostic field, Fassy et al reported a microfluidic qPCR capable of processing 192 samples in parallel and quantifying the expression of different SARS-CoV-2 viral genes [ 23 ], whereas for serological testing, Swank et al developed a microfluidic immunoassay for the detection of anti-SARS-CoV-2 IgG antibodies with the capacity to analyze several samples using a minimal volume of reagents [ 24 ].…”