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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Public Library of Science (PLoS)
2021
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oai:doaj.org-article:152a04f7822c4e649909b2a6062dac582021-12-02T20:18:55ZVersatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples.1932-620310.1371/journal.pone.0243333https://doaj.org/article/152a04f7822c4e649909b2a6062dac582021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0243333https://doaj.org/toc/1932-6203The 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.Julien FassyCaroline LacouxSylvie LeroyLatifa NoussairSylvain HubacAurélien DegoutteGeorges VassauxVianney LeclercqDavid RouquiéCharles-Hugo MarquetteMartin RottmanPatrick TouronAntoinette LemoineJean-Louis HerrmannPascal BarbryJean-Louis NahonLaure-Emmanuelle ZaragosiBernard MariPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 4, p e0243333 (2021) |
| institution |
DOAJ |
| collection |
DOAJ |
| language |
EN |
| topic |
Medicine R Science Q |
| spellingShingle |
Medicine R Science Q Julien Fassy Caroline Lacoux Sylvie Leroy Latifa Noussair Sylvain Hubac Aurélien Degoutte Georges Vassaux Vianney Leclercq David Rouquié Charles-Hugo Marquette Martin Rottman Patrick Touron Antoinette Lemoine Jean-Louis Herrmann Pascal Barbry Jean-Louis Nahon Laure-Emmanuelle Zaragosi Bernard Mari Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples. |
| description |
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. |
| format |
article |
| author |
Julien Fassy Caroline Lacoux Sylvie Leroy Latifa Noussair Sylvain Hubac Aurélien Degoutte Georges Vassaux Vianney Leclercq David Rouquié Charles-Hugo Marquette Martin Rottman Patrick Touron Antoinette Lemoine Jean-Louis Herrmann Pascal Barbry Jean-Louis Nahon Laure-Emmanuelle Zaragosi Bernard Mari |
| author_facet |
Julien Fassy Caroline Lacoux Sylvie Leroy Latifa Noussair Sylvain Hubac Aurélien Degoutte Georges Vassaux Vianney Leclercq David Rouquié Charles-Hugo Marquette Martin Rottman Patrick Touron Antoinette Lemoine Jean-Louis Herrmann Pascal Barbry Jean-Louis Nahon Laure-Emmanuelle Zaragosi Bernard Mari |
| author_sort |
Julien Fassy |
| title |
Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples. |
| title_short |
Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples. |
| title_full |
Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples. |
| title_fullStr |
Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples. |
| title_full_unstemmed |
Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples. |
| title_sort |
versatile and flexible microfluidic qpcr test for high-throughput sars-cov-2 and cellular response detection in nasopharyngeal swab samples. |
| publisher |
Public Library of Science (PLoS) |
| publishDate |
2021 |
| url |
https://doaj.org/article/152a04f7822c4e649909b2a6062dac58 |
| work_keys_str_mv |
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