Samples were vigorously vortexed, and from each elution 200 μL swab extraction were processed using RNAdvance Viral kit (Beckman Coulter) on the Biomek i7 Automated Workstation (Beckman Coulter), according to the manufacturer's protocol. Each sample was eluted in 50 μL of RNase-free water. Real-time RT-PCR assays, targeting the SARS-CoV-2 E gene, were performed using the SensiFAST Probe Lo-ROX One-Step kit (Bioline). The final concentration of primers was 600 nM and the probe concentration was 300 nM. Primers and probe for the E gene assay were taken from the Berlin protocol published in the WHO recommendation for the detection of SARS-CoV-2. Thermal cycling was performed at 48°C for 20 min for reverse transcription, followed by 95°C for 2 min, and then 45 cycles of 94°C for 15 s, 60°C for 35 s. Virus infectivity was tested by seeding quadruplets of 200 μL on VERO E6 cells for CPE assay as described above. The limit of detection of the CPE assay has been determined to be 10 pfu/mL.
Biomek i7 automated workstation
Manufactured by Beckman Coulter
Sourced in United States
The Biomek i7 Automated Workstation is a liquid handling system designed for automated sample preparation and assay workflow. It features precise liquid handling capabilities and a modular design to accommodate a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Rnadvance viral kit, by Beckman Coulter (2 mentions)
Sensifast probe lo rox one step kit, by Meridian Bioscience (2 mentions)
Qubit dsdna hs kit, by Thermo Fisher Scientific (1 mentions)
Hs ngs fragment kit, by Agilent Technologies (1 mentions)
Standard sensitivity rna kit, by Agilent Technologies (1 mentions)
Fragment analyzer automated ce system, by Agilent Technologies (1 mentions)
Agencourt rnadvance blood kit, by Beckman Coulter (1 mentions)
Biomek fxp laboratory automation workstation, by Beckman Coulter (1 mentions)
Quant it ribogreen rna assay kit, by Thermo Fisher Scientific (1 mentions)
Universal plus mrna seq kit, by Tecan (1 mentions)
Rnadvance viral reagent kit, by Beckman Coulter (1 mentions)
Quantstudio 6 system, by Thermo Fisher Scientific (1 mentions)
Floqswab, by Copan (1 mentions)
D300e digital dispenser, by Tecan (1 mentions)
Sensifasttm probe lo rox one step kit, by Meridian Bioscience (1 mentions)
Rnadvance viral xp kit, by Beckman Coulter (1 mentions)
Primer express software, by Thermo Fisher Scientific (1 mentions)
6 protocols using biomek i7 automated workstation
1
SARS-CoV-2 Detection by Real-Time RT-PCR
2
RNA Extraction and RNA-seq Library Prep
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Total RNA from PAXgene preserved blood was extracted using the Agencourt RNAdvance Blood Kit (Beckman Coulter, Indianapolis, IN) on a BioMek FXP Laboratory Automation Workstation (Beckman Coulter). Concentration and integrity (RIN) of isolated RNA were determined using the Quant‐iT™ RiboGreen™ RNA Assay Kit (Thermo Fisher) and an RNA Standard Sensitivity Kit (DNF‐471, Agilent Technologies, Santa Clara, CA, USA) on a Fragment Analyzer Automated CE system (Agilent Technologies), respectively. Subsequently, cDNA libraries were constructed from total RNA using the Universal Plus mRNA‐Seq kit (Tecan Genomics, San Carlos, CA, USA) in a Biomek i7 Automated Workstation (Beckman Coulter). Briefly, mRNA was isolated from purified 300 ng total RNA using oligo‐dT beads and used to synthesize cDNA following the manufacturer's instructions. The transcripts for ribosomal RNA (rRNA) and globin were further depleted using the AnyDeplete kit (Tecan Genomics) prior to the amplification of libraries. Library concentration was assessed fluorometrically using the Qubit dsDNA HS Kit (Thermo Fisher), and quality was assessed with the HS NGS Fragment Kit (1–6,000 bp; DNF‐474, Agilent Technologies).
3
SARS-CoV-2 RNA Detection from Naso/Oropharyngeal Swabs
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Naso/oropharyngeal swab samples (FLOQSwab; Copan, Brescia, Italy) for detection of SARS-CoV-2 RNA were collected in 3 mL sterile universal transport medium (UTM; Copan) and stored at 4 °C for up to four days prior to testing. Viral RNA was extracted from 200 µL sample using the RNAdvance Viral Reagent Kit (Beckman Coulter Inc., Brea, CA, USA) in a Biomek i7 Automated Workstation (Beckman Coulter Inc.), according to the manufacturer’s instructions. Real-time reverse transcriptase PCR was performed in a QuantStudio 6 system (Applied Biosystems, Beijing, China) targeting the envelope (E) gene specific to the Sarbecovirus [17 (link)]. The limit of detection was 20 viral RNA genome copies/mL. The results were reported as positive (Ct values ≤ 40) or negative (Ct values > 40).
4
SARS-CoV-2 Detection Using RT-qPCR
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All procedures for RNA extraction and RT real-time PCR analysis were done as described previously [15 ]. Briefly, 200 µl of each sample was transferred to 150 µl of lysis buffer, followed by incubation for 20 minutes at room temperature for virus denaturation and inactivation. RNA was extracted using an RNAdvance Viral Kit and a Biomek i7 Automated Workstation (Beckman Coulter) and eluted with 25-50 µl of water.
Primers and probes for the SARS-CoV-2 E and N genes were designed according to the Berlin protocol [16 ], and RT-PCR assays were performed according to WHO instructions, using a SensiFAST Probe Lo-ROX One-Step Kit (Bioline). The sensitivity of the RT real-time PCR was determined by performing a virus dilution assay, and the lowest virus concentration detected was 0.1 pfu/ml (Supplementary Fig. S1).
Primers and probes for the SARS-CoV-2 E and N genes were designed according to the Berlin protocol [16 ], and RT-PCR assays were performed according to WHO instructions, using a SensiFAST Probe Lo-ROX One-Step Kit (Bioline). The sensitivity of the RT real-time PCR was determined by performing a virus dilution assay, and the lowest virus concentration detected was 0.1 pfu/ml (Supplementary Fig. S1).
5
High-Throughput Drug Screening Protocol
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High-throughput drug screens were conducted in collaboration with the high-throughput screening facility of the Princess Máxima Center [16 ]. The screens were performed with 384-well plates and a library containing 198 drugs using the high-throughput screening facility (Beckman Coulter with a Biomek i7 Automated Workstation). Using the Echo 550 dispenser, the drugs (in DMSO or MQ, at different concentrations) were added to the wells containing the cells, at final concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM and 10 μM (0.25% DMSO or MQ). Combination validation screens were conducted using a 10 × 10 matrix of five-fold concentration ranges from 0.03 nM to 10 μM (0.25% DMSO) using the D300e Digital Dispenser (TECAN). For all screens, cells treated with DMSO were used as positive controls and for the high-throughput screens, cells treated with staurosporine (final concentration of 10 μM) were used as negative controls. Supplementary Table 2 summarizes the compounds included in this study.
6
SARS-CoV-2 Viral RNA Detection in Nursing Homes
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Negative and positive qRT-PCR nasopharyngeal-swab samples from symptomatic and asymptomatic individuals were collected as part of routine scanning of nursing homes. Ethical review and approval were waived, since the samples used for this study were leftovers of anonymized samples. No information is available on the level of symptoms manifested by each tested positive individual. Viral RNA was extracted using RNAdvance Viral XP kit (Beckman Coulter). From each sample 200 μL were added to LBF lysis buffer, and further processed on the Biomek i7 Automated Workstation (Beckman Coulter), according to the manufacturer's protocol. Each sample was eluted in 50 μL of RNase-free water. Real-time RT-PCR assays were performed using the SensiFASTTM Probe Lo-ROX one-step kit (Bioline). In each reaction the primers final concentration was 600 nM and the probe concentration was 300 nM. Thermal cycling was performed at 48°C for 20 minutes for reverse transcription, followed by 95°C for 2 min, and then 45 cycles of 94°C for 15 sec, 60°C for 35 sec. Primers and probes (listed in Supplementary Table S2) were designed using the Primer Express Software (Applied Biosystems) and purchased from Integrated DNA Technologies, Inc.
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