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

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. The...

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Veröffentlicht in:PloS one 2021-04, Vol.16 (4), p.e0243333-e0243333
Hauptverfasser: Fassy, Julien, Lacoux, Caroline, Leroy, Sylvie, Noussair, Latifa, Hubac, Sylvain, Degoutte, Aurélien, Vassaux, Georges, Leclercq, Vianney, Rouquié, David, Marquette, Charles-Hugo, Rottman, Martin, Touron, Patrick, Lemoine, Antoinette, Herrmann, Jean-Louis, Barbry, Pascal, Nahon, Jean-Louis, Zaragosi, Laure-Emmanuelle, Mari, Bernard
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Sprache:eng
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Zusammenfassung: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0243333