SARS-CoV-2 Detection for Diagnosis Purposes in the Setting of a Molecular Biology Research Lab

Coupeau, Damien; Burton, Nicolas P.; Lejeune, Noémie; Loret, Suzanne; Petit, Astrid; Pejaković, Srđan; Poulain, Florian; Bonil, Laura; Trozzi, Gabrielle; Wiggers, L.; Willemart, Kévin; André, Emmanuel; Laenen, Lies; Cuypers, Lize; Ranst, Marc Van; Bogaerts, Pierre; Muylkens, Benoît; Gillet, Nicolas

doi:10.3390/mps3030059

Cited by 13 publications

(17 citation statements)

References 10 publications

(14 reference statements)

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“…Total RNAs (1 µg), including viral and human RNAs, were reverse-transcribed using the Super Script II RNase H reverse transcriptase kit according to manufacturer’s instructions (Invitrogen, Merelbeke, BE). cDNA was used to amplify SARS-CoV-2 gene E with Takyon Taqman kit and human genes with Takyon SYBR Green kit (Eurogentec, Liège, BE) in a Light Cycler 96 device (Roche Diagnostics, Mannheim, DE) ( Coupeau et al, 2020 ). Primer sequences are listed in Table 1 .…”

Section: Methodsmentioning

confidence: 99%

Susceptibility of neuroblastoma and glioblastoma cell lines to SARS-CoV-2 infection

et al. 2021

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Modelling cell infection in-a-dish can represent a useful tool to understand the susceptibility of different cell types towards severe acute respiratory coronavirus-2 (SARS-CoV-2) and to decipher its neurotropism. In this perspective, retinoic acid (RA)-differentiated neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2) and glioblastoma cell lines, U-87 MG and U-373 MG, were infected with a SARS-CoV-2 strain, at various multiplicity-of-infection (MOI). We first demonstrated that the common entry genes – needed for invading epithelial cells – were expressed. RA-differentiation induced an upregulation of ace2 and tmprss2 gene expression while inducing downregulation of ctsb and ctsl . Using in situ hybridization and confocal analysis, SARS-CoV-2 gene S RNA was detected intracellularly at MOI 5.0, and localized in both soma and neuritic-like or glial-like processes. The infection was confirmed by quantification of viral gene E RNA and showed a dose-dependency, with few infected cells at MOI 0.1. After 24 h of infection, no cytopathic effect was observed in SH-SY5Y abilities to maintain neuritic processes or in U-373 MG for the uptake of glutamate. Unlike the permissive Vero E6 cells, no significant apoptosis death was detected following SARS-CoV-2 infection of neuroblastoma or glioblastoma cells. This study demonstrates the susceptibility of neuronal- and glial-like cell lines towards SARS-CoV-2 infection at high MOIs. Once inside the cells, the virus does not seem to rapidly replicate nor exert major cytopathic effect. Overall, our results strengthen the idea that SARS-CoV-2 has a tropism for nervous cells that express commonly described entry genes.

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Section: Methodsmentioning

confidence: 99%

Susceptibility of neuroblastoma and glioblastoma cell lines to SARS-CoV-2 infection

et al. 2021

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“…However, false negative results due to inappropriate sample collection timing, sample type, sampling technique or other problems have limited its usage and thus increased the importance of serological testing (Coupeau et al . 2020 ; Lee et al . 2020 ; Li and Ren 2020 ; Li H et al .…”

Section: Discussionmentioning

confidence: 99%

Summary of the Detection Kits for SARS-CoV-2 Approved by the National Medical Products Administration of China and Their Application for Diagnosis of COVID-19

Ruhan

Wang

et al. 2020

Virol. Sin.

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The on-going global pandemic of coronavirus disease 2019 (COVID-19) caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been underway for about 11 months. Through November 20, 2020, 51 detection kits for SARS-CoV-2 nucleic acids (24 kits), antibodies (25 kits), or antigens (2 kits) have been approved by the National Medical Products Administration of China (NMPA). Convenient and reliable SARS-CoV-2 detection assays are urgently needed worldwide for strategic control of the pandemic. In this review, the detection kits approved in China are summarised and the three types of tests, namely nucleic acid, serological and antigen detection, which are available for the detection of COVID-19 are discussed in detail. The development of novel detection kits will lay the foundation for the control and prevention of the COVID-19 pandemic globally.

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“…Reactions to detect SARS-CoV-2 E sequence were performed in 25 μL as described by Coupeau et al . [5]. Briefly, a 25 μL reaction contained RNAse free water, 5 μL 5X concentrated reaction master mix containing DNA polymerase, MgCl 2 and dNTP (Eurogentec Takyon One-Step No Rox Probe 5X MasterMix dTTP), 0.25 μL Euroscript II reverse transcriptase and RNAse inhibitor and 0.25 μL additive (both provided with the Eurogentec Takyon 5X MasterMix), 5 μL RNA and various concentration of primers and probes.…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, a 25 μL reaction contained RNAse free water, 5 μL 5X concentrated reaction master mix containing DNA polymerase, MgCl 2 and dNTP (Eurogentec Takyon One-Step No Rox Probe 5X MasterMix dTTP), 0.25 μL Euroscript II reverse transcriptase and RNAse inhibitor and 0.25 μL additive (both provided with the Eurogentec Takyon 5X MasterMix), 5 μL RNA and various concentration of primers and probes. In singleplex assays, primers and probes (targeting either the SARS-CoV-2 E or the Schmallenberg virus RNA) were at 400 nM and 200 nM final concentration, respectively, as previously described [5]. In duplex assays, the working concentration of primers and probe targeting the SARS-CoV-2 E sequence were at 100 nM and 50 nM, respectively, and the working concentration of primers and probe targeting the Schmallenberg virus RNA internal control were at 200 nM and 100 nM, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Rapidly, the additional support of fundamental research laboratories allowed to develop quality controlled diagnosis workflows. In Belgium, a veterinary research unit of the University of Namur (NARILIS) developed and provides an internal control, RNA extract containing Schmallenberg virus RNA genome, for each RT-qPCR reaction [5]. SARS-CoV-2 RT-qPCR are consequently only validated when a parallel RT-qPCR targeting the Schmallenberg virus RNA genome show positive results.…”

Section: Introductionmentioning

confidence: 99%

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Quality controlled SARS-CoV-2 duplex procedure to reduce time and scarce molecular biology diagnosis reagents

Collin¹,

Chaboteaux²,

Fontaine

et al. 2020

Preprint

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The aim of this study was to provide validated procedures allowing to detect the SARS-CoV-2 and an internal control in a unique one-step duplex RT-qPCR. Two internal controls were tested, targeting either the Schmallenberg virus RNA provided by the NARILIS laboratory (University of Namur) with a HEX-labelled probe or a Diagenode Diagnostics internal control with a Cy5-labelled probe. Our results showed that Ct values of the RT-qPCR duplex assay were even smaller in the optimized working conditions, allowing to use the optimized qPCR conditions in routine diagnosis.

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SARS-CoV-2 Detection for Diagnosis Purposes in the Setting of a Molecular Biology Research Lab

Cited by 13 publications

References 10 publications

Susceptibility of neuroblastoma and glioblastoma cell lines to SARS-CoV-2 infection

Susceptibility of neuroblastoma and glioblastoma cell lines to SARS-CoV-2 infection

Summary of the Detection Kits for SARS-CoV-2 Approved by the National Medical Products Administration of China and Their Application for Diagnosis of COVID-19

Quality controlled SARS-CoV-2 duplex procedure to reduce time and scarce molecular biology diagnosis reagents

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