期刊
PLOS ONE
卷 16, 期 4, 页码 -出版社
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0243333
关键词
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资金
- Delegation CNRS Cote d'Azur
- UCA GenomiX platform and MICA imaging facility of the University Cote d'Azur
- Propagate consortium
- Centre National de la Recherche Scientifique (CNRS)
- Universite Cote d'Azur
- French French Defence Innovation Agency - Agence de l'Innovation de Defense
- Departement des Alpes Maritimes
- Canceropole PACA
- Plan Cancer 2018 ARN noncodants en cancerologie: du fondamental au translationnel [18CN045]
- Cote d'Azur-Nice-Interdisciplinary Institute for Artificial Intelligence [ANR-19-P3IA-0002-3IA]
- France Genomique [ANR-10-INBS-6 09-03, ANR-10-INBS-09-02]
The emergence and fast spread of SARS-CoV-2 has highlighted the insufficient testing capacity in many countries due to limitations in resources, technology, and personnel. While traditional RT-qPCR diagnostic tests remain the standard method, a quantitative nanofluidic assay based on the Biomark™ instrument from Fluidigm offers a more efficient and cost-effective approach to testing, overcoming some of the limitations of traditional methods. This advanced system allows for high-throughput testing, monitoring of specific SARS-CoV-2 variants, and detection of other pathogens and host cellular responses.
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 Biomark(TM) 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 Biomark(TM) 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.
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