Extraction of cell free DNA from plasma was performed using a fully automated QIAGEN platform, QIAsymphony SP, and QIAsymphony DSP Virus/Pathogen Midi Kit following centrifugation. Sequence libraries were prepared according to the KAPA Hyper protocol (Kapa Biosystems) with the ligation of Illumina sequence adaptors followed by PCR amplification and clean-up. Barcoded libraries were hybridized with DNA probes targeting all coding exons of ERBB2 (Integrated DNA Technologies) in two successive captures, using a protocol modified from the NimbleGen SeqCap Target Enrichment system. The first capture was incubated at 55°C for 16 h followed by post-capture washes and 16 cycles of PCR amplification. The second capture was incubated at 65°C for 4 h followed by post-capture washes and 3–5 cycles of PCR amplification. Captured libraries were sequenced on an Illumina HiSeq as paired-end 100 bp reads.
Dsp virus pathogen midi kit
Manufactured by Qiagen
Sourced in Germany
The DSP Virus/Pathogen Midi Kit is a laboratory product designed for the isolation and purification of viral and pathogenic nucleic acids from various sample types. The kit utilizes a spin column-based technology to efficiently extract and concentrate target nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Qiasymphony, by Qiagen (5 mentions)
Qiasymphony sp, by Qiagen (3 mentions)
Proteinase k, by Qiagen (2 mentions)
Tissue lysis solution, by Qiagen (2 mentions)
Lysozyme, by Merck Group (2 mentions)
Lysostaphin, by Merck Group (2 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase and rnaseout, by Thermo Fisher Scientific (2 mentions)
Ion library taqman quantitation kit, by Thermo Fisher Scientific (1 mentions)
Ion 16s metagenomics kit, by Thermo Fisher Scientific (1 mentions)
Ion xpress plus fragment library kit, by Thermo Fisher Scientific (1 mentions)
Ion s5 sequencer, by Thermo Fisher Scientific (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Realtime hiv 1 assay, by Abbott (1 mentions)
Qiasymphony platform, by Qiagen (1 mentions)
Lightcycler 2.0 platform, by Roche (1 mentions)
Qiasymphony sp as platform, by Qiagen (1 mentions)
Realstar sars cov 2 rt pcr kit 1, by Altona Diagnostics (1 mentions)
Allplex 2019 ncov assay, by Seegene (1 mentions)
Qiasymphony dsp dna mini kit, by Qiagen (1 mentions)
17 protocols using dsp virus pathogen midi kit
1
Targeted Sequencing of ERBB2 from cfDNA
2
Quantitative CMV DNA Extraction and Detection
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Clinical specimens obtained from the patients after conventional carotid endarterectomy were first subjected to physical disintegration with the help of a scalpel in sterile petri dishes. After the procedure, the shredded clinical specimens were placed in 20 mg sterile Eppendorf tubes with 400 μl of tissue lysis solution (Qiagen, Cat no: 939011), and 40 μl of proteinase K (Qiagen, Cat no: 19133) was added, in accordance with the kit procedure, to perform overnight incubation at 56°C. Then, 600 μl of AVE solution was added into the Eppendorf tubes containing the clinical specimens, and CMV DNA extraction was performed. Extraction of the samples was performed using the DSP Virus/Pathogen midi kit (Qiagen, Cat no. 937055) on the Qiasymphony SP/AS platform. PCR was performed using the Artur CMV QS-RGQ kit (24) (Cat no: 4503363) on a Rotorgene Q instrument. Assessment of the results was based on the Rotorgene Q device in the green channel showing the presence of CMV DNA in clinical specimens and in the yellow channel displaying the internal control for any inhibition in the study.
3
Microbial Profiling from Nasal/Oropharyngeal Swabs
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Nasal/oropharyngeal swabs were collected from patients and Microbial DNA was extracted from 350 µL of samples using the QIAsymphony automatic extractor with DSP Virus/Pathogen Midi Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. 16S gene hypervariable regions were amplified using two separate pools of primers targeting V2–4–8 and V3–6 and 7–9 regions with the Ion 16S Metagenomics Kit (ThermoFisher, Milan; Italy). Amplification was performed separately for the two pools of primer for each sample, then the two reactions tubes were pooled and used for library preparation in the following steps. The Ion Xpress Plus Fragment Library Kit (ThermoFisher, Milan; Italy) was used following the manufacturer’s instructions. Produced libraries were then purified using the AMPure XP Beads (Beckman Coulter, Milan; Italy) and quantified using the Ion Library TaqMan Quantitation Kit (ThermoFisher, Milan; Italy) on the 7900 instrument (Applied Biosystem) and the High-Sensitivity DNA Kit on Bioanalyzer 3100 (Agilent, Santa Clara (CA) United States). Sequencing was performed on an Ion 530 chip using the Ion S5 Sequencer (ThermoFisher, Milan; Italy) obtaining, on average, 5 × 105 reads per sample (range: 3 × 105–9 × 105).
4
SARS-CoV-2 Detection from Clinical Samples
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If there were remnants of original tissue/swab, specimen RNA was extracted out of the remaining original sample prepared for virus isolation using the QIAsymphony instrument (DSP Virus/Pathogen Midi Kit; Qiagen; Hilden, Germany). SARS-CoV-2 RNA was detected by using the AllplexTM 2019-nCoV (Seegene; Seoul, Korea) according to the manufacturer’s recommendations.
5
Ultrasensitive HIV-1 Viral Load and DNA Quantification
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Residual viral load (RVL) was quantified with the ultrasensitive protocol of the Abbott RealTime HIV-1 assay (LLOD: 5 copies/ml), obtained applying the following modifications to the standard procedure (19 (link)): (1) higher sample volume (3.2 ml) concentrated by ultracentrifugation; (2) calibration curve extended toward HIV-RNA lower levels; (3) reduced volume of internal control; (4) “open” software. Each patient underwent multiple quantifications of RVL.
Total HIV-DNA was quantified in PBMCs collected by density gradient centrifugation as previously reported. Automated DNA extraction from PBMC was performed on the QIAsymphony platform using an inhouse modification of the DSP Virus/Pathogen Midi Kit protocol (Qiagen, Milan Italy). A Real-Time PCR targeting HIV-LTR was used to quantify total HIV-1 DNA as previously described (20 (link)), whereas another Real-Time PCR targeting the housekeeping gene hTERT was performed to refer HIV-DNA copies to a million PBMCs (21 (link)) on LightCycler 2.0 platform (Roche, Monza Italia).
Total HIV-DNA was quantified in PBMCs collected by density gradient centrifugation as previously reported. Automated DNA extraction from PBMC was performed on the QIAsymphony platform using an inhouse modification of the DSP Virus/Pathogen Midi Kit protocol (Qiagen, Milan Italy). A Real-Time PCR targeting HIV-LTR was used to quantify total HIV-1 DNA as previously described (20 (link)), whereas another Real-Time PCR targeting the housekeeping gene hTERT was performed to refer HIV-DNA copies to a million PBMCs (21 (link)) on LightCycler 2.0 platform (Roche, Monza Italia).
6
CMV DNA Extraction and Quantification
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The samples taken from patients after classical phlebectomy were subjected to physical fragmentation. After the fragmentation, 20-mg samples were placed in 2-ml sterile Eppendorf tubes, and 40 μl of tissue lysis solution (Qiagen, cat no. 939011) and 40 μl of proteinase K (Qiagen, cat no. 19133) were added and incubated at 56°C overnight. Then, 600 μl of AVE solution was added to the Eppendorf tubes and passed to the CMV DNA extraction phase. Extraction was performed on a QIAsymphony SP/AS platform using a DSP Virus/Pathogen midi kit (Qiagen, cat no. 937055). PCR was performed on Rotorgene Q using an Artus CMV QS-RGQ kit (24) (cat no. 4503363). During evaluation, CMV DNA existence in the clinical samples was observed at the green channel and the existence of any inhibition was observed in the yellow channel on the Rotorgene Q device.
7
Bacterial DNA Extraction Protocol
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Sample pellets were thawed and a 500 μL combination of lysozyme (20 mg/mL, Sigma-Aldrich, St. Louis MO) and lysostaphin (200 μg/mL, Sigma-Aldrich, St. Louis MO) was applied to chemically lyse the bacterial cell walls. DNA extraction was then performed using the DSP Virus/Pathogen Midi Kit and the Complex800_V6_DSP protocol on the QIAsymphony SP (Qiagen, Valencia CA).
8
SARS-CoV-2 Detection from Clinical Specimens
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All self-collected specimens were tested at the Institute of Medical Virology, Goethe University Frankfurt. Dry swabs (nasal swab, tongue swab) were suspended in 2 mL of phosphate-buffered saline (PBS) and incubated for 5 min prior to further processing. Depending on the viscosity in the self-collected specimen and to achieve the required input volume, saliva was diluted up to 1:2.5. The chewed cotton pads with saliva were suspended in 4 mL of PBS, compressed, and incubated for 5 min. Gargle solution was used native, without further dilution. Samples were stored at −80 °C until nucleic acid extraction and PCR-based testing was performed. Specimens were extracted using the QIAsymphony and the DSP virus/pathogen midi kit (both Qiagen GmbH, Hilden, Germany) according to the manufacturers’ protocols. Realtime RT-PCR (rRT-PCR) analysis was performed using the RealStar® SARS-CoV-2 RT-PCR Kit 1.0 (Altona Diagnostics GmbH, Hamburg, Germany) [17 (link)] on the ABI PRISM® 7500 Analyser (Applied Biosystems, Waltham, MA, USA) according to the manufacturers´ specifications. Three quantitative comparison samples containing 105, 106, and 107 SARS-CoV-2 (BetaCoV/Munich/ChVir984/2020) RNA copies/mL were used to generate a standard curve and to calculate the viral RNA copies/mL. Equivocal test results were excluded from the analysis of testing sensitivity.
9
SARS-CoV-2 RNA Extraction and Detection
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Left over material from patient samples (dry swabs suspended in 1 mL phosphate buffered saline (PBS)) was diluted 1:5 in PBS in order to gain enough sample material for further testing.
When nucleic acid extraction was required (i.e. Allplex™ 2019-nCoV Assay (Seegene Inc., Seoul, South Korea) and the RealStar® SARS-CoV-2 RT-PCR Kit 1.0 (altona Diagnostics GmbH, Hamburg, Germany)), 500 μL of each sample was extracted using the QIAsymphony (Qiagen GmbH, Hilden, Germany) together with the DSP virus/pathogen midi kit (Qiagen) according to manufacturers’ instructions and eluted in a final volume of 130 μL. After extraction, the nucleic acid was stored at −80 °C until further testing.
When nucleic acid extraction was required (i.e. Allplex™ 2019-nCoV Assay (Seegene Inc., Seoul, South Korea) and the RealStar® SARS-CoV-2 RT-PCR Kit 1.0 (altona Diagnostics GmbH, Hamburg, Germany)), 500 μL of each sample was extracted using the QIAsymphony (Qiagen GmbH, Hilden, Germany) together with the DSP virus/pathogen midi kit (Qiagen) according to manufacturers’ instructions and eluted in a final volume of 130 μL. After extraction, the nucleic acid was stored at −80 °C until further testing.
10
DNA Extraction and Sequencing Protocol
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DNA was prepared from whole blood with a simple salting-out method [21 (link)], or (for the Thai screening cohort) with QIAsymphony DSP DNA Mini Kit from QIAGEN (Venlo, Netherlands). DNA from plasma samples was also extracted with QIAsymphony but using the DSP virus/pathogen Midi Kit from QIAGEN according to the manufacturer's instructions. Amplification was done as previously described [22 (link)] using Expand high-fidelity PCR system (Roche, Basel, Switzerland), with some modifications. In brief, 40.5–100 ng DNA was added to a 20 μL reaction mix of 0.2 U Taq polymerase, 6.7 pmol of each primer (Table S1 ) and 2 nmol dNTP mix. Reactions in 35 thermal cycles (after 3 min, 95 °C): 95 °C (20 s), 58 °C (30 s), 72 °C (40 s or 3 min, depending on amplicon size). Amplicons were purified in 3% agarose gel and extracted in nuclease free H2O with gel extraction kits QIAquick (QIAGEN) and GeneJET (Thermo Scientific, Vilnius, Lithuania). Sanger sequencing was executed by Eurofins Genomics (Ebersberg, Germany), or in house as previously described [22 (link)] with primers stated in Table S1 .
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