Versatile and flexible microfluidic qPCR test for high-throughput SARS-CoV-2 and cellular response detection in nasopharyngeal swab samples
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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 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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SciScore for 10.1101/2020.11.09.20228437: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Statistical analysis: Statistical analyses were performed using GraphPad Prism software. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:An important limitation of current tests is the requirement for …
SciScore for 10.1101/2020.11.09.20228437: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Statistical analysis: Statistical analyses were performed using GraphPad Prism software. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:An important limitation of current tests is the requirement for RNA extraction that constitutes an obstacle to scale-up the capacity of testing both in term of time and cost. Several groups have explored methods to circumvent RNA extraction by performing RT-qPCR directly on crude or heat-inactivated clinical samples (8–10). Overall these methods show that testing for SARS-CoV-2 infection can be performed without RNA extraction, with a limited loss in accuracy for determining negative and positive cases. While this procedure is simple and attractive, it might be improved by the addition of detergents to facilitate viral capsid lysis to release genomic RNA and also directly inactivate the virus to facilitate sample handling and safety. Several methods and commercial kits have developed approaches to lyse efficiently mammalian cells and directly perform RT-qPCR or RNA-seq libraries. However, these methods are not fully optimized for virus lysis, which requires increased concentrations of detergents. Some studies indicate that Triton X-100, widely used in virus inactivation procedures, or Tween-20 may slightly improve or at least not interfere with the RT-qPCR SARS-CoV-2 direct testing of nasopharyngeal swabs or saliva (9, 16). However, these initial reports clearly mentioned that additional efforts were needed to optimize direct RT-qPCR assays on detergent-inactivated samples. We evaluated here the use of several detergents and emulsifiers (Triton X-100, Tween-20, Brij™-35, Brij...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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