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

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 couple of probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction steps. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the Biomark 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 in addition to SARS-CoV-2 probes of other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). Its 10 nL range volume 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 procedures, 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.

An international, interlaboratory ring trial confirms the feasibility of an extraction-less “direct” RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples

“…The opportunity and feasibility described here is not simply theoretical. At the time of publication, several of the Propagate Network partner laboratories (Brazil, France, United States) are promoting or exploring the broad-scale adoption and implementation of this method for ongoing SARS-CoV-2 public health screening efforts in their regions [ 20 ]. Additionally, in October 2020, the Infectious Disease Diagnostic Laboratory at the Children’s Hospital of Philadelphia implemented an extraction-free protocol for routine diagnostic testing of SARS-CoV-2 [ 21 ].…”

An international, interlaboratory ring trial confirms the feasibility of an open-source, extraction-less “direct” RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples

“…The opportunity and feasibility described here is not simply theoretical. At the time of publication, several of the Propagate Network partner laboratories (Brazil, France, United States) are promoting or exploring the broad-scale adoption and implementation of this method for ongoing SARS-CoV-2 public health screening efforts in their regions [19]. Additionally, in October 2020, the Infectious Disease Diagnostic Laboratory at the Children’s Hospital of Philadelphia implemented an extraction-free protocol for routine diagnostic testing of SARS-CoV-2 ( personal communication ).…”