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.
Highlights
To control the pandemic and monitor virus propagation of SARS-CoV-2 in real time, extensive testing is necessary
We evaluated the use of a high-throughput real-time PCR device, the BiomarkTM HD, to increase throughput, flexibility in probe inclusion, and decrease reagent consumption, together with an optimized protocol for SARS-CoV-2 RNA detection without RNA extraction
This system requires a pre-amplification step after cDNA synthesis. This is due to the low volume of the integrated fluidic circuits (IFCs) reaction chamber (9nl)
Summary
To control the pandemic and monitor virus propagation of SARS-CoV-2 in real time, extensive testing is necessary. Alternatives are available [1, 2], viral load detection from nasopharyngeal or saliva samples is still the most appropriate method to identify SARS-CoV-2 carriers. The current diagnostic testing methods recommended by the Centers for Diseases Control (CDC) and the World Health Organization (WHO) are based on a traditional RTqPCR assay, with validated primers [3]. The availability of this assay has been a major hurdle in the orderly and efficient management of the pandemic because of reagent shortages, as well as material and staff limitations. To face material shortage and reduce processing times, two kinds of process optimization should be undertaken: (1) multiplexing sample and PCR probes, as well as (2) reducing the steps in sample preparation
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