RESUMO
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.
Assuntos
COVID-19/diagnóstico , Técnicas Analíticas Microfluídicas/métodos , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodos , Adulto , COVID-19/virologia , Teste para COVID-19/métodos , Primers do DNA , Testes Diagnósticos de Rotina/métodos , Feminino , Humanos , Masculino , MicroRNAs/genética , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Sensibilidade e EspecificidadeRESUMO
The Forensic Science Institute of the French "Gendarmerie Nationale" (IRCGN™) developed in 2015 an ISO 17025 certified mobile DNA laboratory for genetic analyses. This Mobil'DNA laboratory is a fully autonomous and adaptable mobile laboratory to perform genetic analyses in the context of crime scenes, terrorism attacks or disasters. To support the hospital task force in Paris during the peak of the COVID-19 epidemic, we adapted this mobile genetic laboratory to perform high-throughput molecular screening for coronavirus SARS-CoV-2 by real-time PCR. We describe the adaptation of this Mobil'DNA lab to assist in Coronavirus SARS-CoV-2 diagnosis.
