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

Abstract

The emergence of the SARS-CoV-2 virus and the exponential growth of COVID-19 cases have created a major crisis for public health systems. The critical identification of contagious asymptomatic carriers requires the isolation of viral nucleic acids, reverse transcription, and amplification by PCR. However, the shortage of specific proprietary reagents or the lack of automated platforms have seriously hampered diagnostic throughput in many countries. Here, we provide a procedure for SARS-CoV-2 detection for diagnostic purposes from clinical samples in the setting of a basic research molecular biology lab. The procedure details the necessary steps for daily analysis of up to 500 clinical samples with a team composed of 12 experienced researchers. The protocol has been designed to rely on widely available reagents and devices, to cope with heterogeneous clinical specimens, to guarantee nucleic acid extraction from very scarce biological material, and to minimize the rate of false-negative results.

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

“…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 .…”

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

“…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 .…”

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

“…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.…”

“…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.…”

“…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.…”