The nasopharyngeal swabs of severe hospitalized and in ICU COVID-19 patients tested positive (n=31) for SARS-CoV-2 by RT-qPCR following the QIAamp Viral RNA Mini or the EZ1 DSP Virus Kits (Qiagen, Hilden, Germany) and the control (qPCR negative) swabs (n=11) were used in the study. Human RNA was extracted from nasopharyngeal swabs using RNeasy 96 QIAcube HT Kit (Qiagen USA) as per the manufacturer’s instructions. 2 ml of Universal Transfer Medium (UTM) was transferred to a clean, sterile Eppendorf tube and centrifuged at 4000 rpm for 3 min. 1650 μl of supernatant was carefully removed without disturbing the pelleted squamous and respiratory epithelial cells. Pellet was resuspended with the remaining 350 μl of Universal Transfer Medium (UTM) and transferred to S Block (RNeasy 96 QIAcube HT Kit, Qiagen USA). Quality and quantity of the extracted RNA was determined using NanoDrop™ 8000 Spectrophotometer (ThermoFisher Scientific, USA) and reverse transcribed to cDNA using a High-Capacity cDNA Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol.
Ez1 dsp virus kit
Manufactured by Qiagen
Sourced in Germany, United States
The EZ1 DSP virus kit is a nucleic acid extraction system designed for the automated purification of viral nucleic acids from various sample types. It utilizes magnetic-bead technology to efficiently capture and extract viral RNA or DNA. The kit is intended for use with the EZ1 Advanced XL instrument to streamline the sample preparation process.
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Lab products found in correlation
Ez1 advanced xl, by Qiagen (5 mentions)
Lightcycler 480, by Roche (4 mentions)
Ez1 advanced xl instrument, by Qiagen (3 mentions)
Ez1 xl advance, by Qiagen (3 mentions)
Ez1 advanced xl system, by Qiagen (3 mentions)
Qiaamp viral rna mini, by Qiagen (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
High capacity cdna kit, by Thermo Fisher Scientific (1 mentions)
Rneasy 96 qiacube ht kit, by Qiagen (1 mentions)
Sureselect xt library preparation kit, by Agilent Technologies (1 mentions)
Novaseq sp reagent kit, by Illumina (1 mentions)
Truseq stranded total rna library kit, by Illumina (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Eswab, by Copan (1 mentions)
Qiacube connect, by Qiagen (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Kingfisher presto system, by Thermo Fisher Scientific (1 mentions)
33 protocols using ez1 dsp virus kit
1
RNA Extraction from COVID-19 Patient Samples
2
Comprehensive SARS-CoV-2 Genomic Profiling
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All 49 COVID-19 patients tested positive for SARS-CoV-2 by RT-qPCR using RNA extracted from nasopharyngeal swabs following the QIAamp Viral RNA Mini or the EZ1 DSP Virus Kits (Qiagen, Hilden, Germany). RNA libraries from all samples were then prepared for shotgun transcriptomic sequencing using the TruSeq Stranded Total RNA Library kit from Illumina (San Diego, CA, USA), following manufacturer’s instructions. Libraries were sequenced using the NovaSeq SP Reagent kit (2 X 150 cycles) from Illumina (San Diego, CA, USA). Sample L5630 underwent a target enrichment approach where double stranded DNA (synthesized using the QuantiTect Reverse Transcription Kit from Qiagen, Hilden, Germany) was amplified using 26 overlapping primer sets covering most of the SARS-CoV-2 genome as recently described by our group9 (link). PCR products were then sheared by ultra-sonication (Covaris LE220-plus series, MA, USA) and prepared for sequencing using the SureSelectXT Library Preparation kit (Agilent, CA, USA). This library was sequenced using the Illumina MiSeq Micro Reagent Kit, V2 (2 X 150 cycles).
3
Automated RNA Extraction for SARS-CoV-2 Testing
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For Standard of Care (SoC) analysis (hereinafter named as Reference method), total RNA extraction was performed using an EZ1® Advanced XL instrument (Qiagen, Hilden, Germany) with EZ1® DSP Virus Kit (Qiagen, Hilden, Germany) according to manufacturer instructions: starting volume was 200 µL, while elution volume was 90 µL. Instead, for the MagMAX™-96 AI/ND Viral RNA Isolation kit adapted to automatic platform Hamilton StarLet (hereinafter named as Testing method), 50 µL of each sample were added to a 96 well plate with U bottom (starting plate), and then the reactive was resuspended according to kit instructions. Afterwards, each component was placed on working desk (Figure 1 a), and extraction protocol was performed as reported in Figure 1 b.
4
Multiplex Respiratory Virus Detection
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The nasopharyngeal swab specimens were collected from the patients at admission using a special swab (ESWAB from Copan Italia SpA) by nursing staff and transported to the laboratory immediately at a storing temperature of 20–25 °C for testing within 1 h of collection. A qualitative nucleic acid multiplex test (The xTAG® Respiratory Viral Panel FAST v2, Luminex) was performed to test the following respiratory viruses: IFV-A, IFV-B, RSV, hCoV, hMPV, PIV-1, PIV-2, PIV-3, and PIV-4, EV/HRV, ADV, and hBoV. Nucleic acid extraction was performed using the EZ1 DSP Virus Kit (Qiagen, Germany).
5
Viral RNA Quantification by RT-qPCR
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Viral RNAs were extracted from 50 μL of serum with the EZ1 apparatus running the EZ1 DSP virus kit (Qiagen). Viral RNA levels were measured by a one-step quantitative reverse transcriptase PCR assay (RT-qPCR) on the Light Cycler 480 (Roche) with primers, probe, and cycling conditions previously described for USUV [68 (link)] and WNV [69 (link)].
6
SARS-CoV-2 Subgenomic RNA Quantification
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Viral RNA was extracted from 200 uL of NP swab and BAL material using the Qiagen EZ1 DSP Virus kit on the automated EZ1 XL Advance instrument (Qiagen, Valencia, CA, USA). RT-qPCR was performed on the 7500 Dx Fast thermal cycler (Thermo Fisher Scientific, Life Technologies, Carlsbad, CA, USA). Amplification and quantification of the sg RNA (targeting the E gene) was performed following the previously described method [22 (link)]. A synthetic RNA for subgenomic E was used as a calibrator. Results were reported in copies/mL.
7
SARS-CoV-2 Detection in Nasopharyngeal Swabs
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Nasopharyngeal swab specimens were taken from all patients and placed in 120–140 µL viral transport media (VTM). RNA was purified from specimens using either the Qiacube Connect (Qiagen), QIAamp Viral RNA mini kit (Qiagen), or the EZ1 DSP Virus kit (Qiagen). SARS-CoV-2 RNA was detected using the qualitative cobas SARS-CoV-2 kit using the cobas 6800 system. For detection, a two-target RT-PCR using (1) SARS-CoV-2-specific primers and (2) pan-sarbecovirus primers as included in the cobas master mix. All assays were performed in a CLIA-certified high complexity laboratory at the Mount Sinai Health System.
8
SARS-CoV-2 RT-PCR Detection Protocol
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The swab specimens were analysed by routine SARS-CoV-2 RT-PCR. Briefly, RNA was extracted from 200µL of eSwabs medium using the automated Microlab Nimbus workstation (Hamilton, Reno, NV, USA) coupled to a Kingfisher Presto system (Thermo Fisher Scientific, Waltham, MA, USA) or using the EZ1 Advanced XL instrument with EZ1 DSP Virus Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
RT-PCR was performed using the Bosphore SARS-CoV-2/Flu/RSV IVD panel (Anatolia geneworks, Sultanbeyli/İstanbul, Turkey), targeting the Orf 1a/b and N genes, using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories S.r.l., Segrate/Mi, Italy). The amplification cycle threshold (Ct) was determined using CFX Maestro (Bio-Rad). Alternatively, the Real-Time PCR SARS-CoV-2 Panel Kit using NeuMoDx istrument (Qiagen Italia, Milan, Italy) was employed, targeting the N and Nsp2 genes. The Ct value for the N target was used as a proxy of the viral load in the corresponding sample. Cellular RnaseP mRNA was used as am endogenous control for the RT-PCR.
RT-PCR was performed using the Bosphore SARS-CoV-2/Flu/RSV IVD panel (Anatolia geneworks, Sultanbeyli/İstanbul, Turkey), targeting the Orf 1a/b and N genes, using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories S.r.l., Segrate/Mi, Italy). The amplification cycle threshold (Ct) was determined using CFX Maestro (Bio-Rad). Alternatively, the Real-Time PCR SARS-CoV-2 Panel Kit using NeuMoDx istrument (Qiagen Italia, Milan, Italy) was employed, targeting the N and Nsp2 genes. The Ct value for the N target was used as a proxy of the viral load in the corresponding sample. Cellular RnaseP mRNA was used as am endogenous control for the RT-PCR.
9
Quantification of Viral RNA Levels
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Organs were crushed with zirconia beads (Fastprep apparatus, MP Biomedicals) in 500 μL PBS before RNA extraction (RNA RNeasy Mini-Kit, Qiagen). Urine and blood RNA were extracted with the EZ1 DSP virus kit (Qiagen), and viral RNA levels were quantified by a one-step quantitative reverse transcriptase PCR assay (RT-qPCR) (Light Cycler 480, Roche) as previously described [46 (link)]. Viral load was reported as TCID50 equivalents per gram using a standard curve produced using serial 10-fold dilutions of USUV with known viral titers.
10
Comparative Viral RNA Extraction Evaluation
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Viral nucleic acids from samples and controls were extracted with two different extraction systems according to the manufacturer's recommendation: (1) one automated, the EZ1® DSP Virus Kit [22] on the EZ1® Advanced XL robot [23] (QIAGEN, Hilden, Germany) (hereinafter QEZ1); (2) another manual, the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Inc., CA, USA) [24] , [25] (hereinafter TFS-MM). For both extraction systems, the following parameters were verified extraction efficiency, repeatability, reproducibility, contamination and concordance.
The extraction efficiency was evaluated through a “relative recovery index”, which provides a relative percentage value of the extraction efficiency between both systems. Based on the GCE value obtained for each theoretical input and the expected GCE value, this recovery index was calculated as follows: RI% = (GCE obtained) / (GCE expected).
The extraction efficiency was evaluated through a “relative recovery index”, which provides a relative percentage value of the extraction efficiency between both systems. Based on the GCE value obtained for each theoretical input and the expected GCE value, this recovery index was calculated as follows: RI% = (GCE obtained) / (GCE expected).
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