Samples were homogenised, then 11 mL were centrifugated at 200,000 x g for 1 hour at 4°C using a XPN80 (Coulter Beckman, Fullerton, United States (US)) ultracentrifuge equipped with a swing rotor (SW41Ti). Viral pellets were resuspended in 200 μL of PBS 1X buffer. The viral concentrate was lysed and extracted using PowerFecal Pro kit (QIAGEN, Hilden, Germany) on a QIAsymphony automated extractor (QIAGEN, Hinden, Germany ), according to a modified manufacturer’s protocol using a larger volume of samples. Extracted nucleic acids were filtered through OneStep PCR inhibitor removal kit (Zymoresearch, Irvine, US), according to the manufacturer’s instructions.
Qiasymphony automated extractor
Manufactured by Qiagen
Sourced in Germany
The QIAsymphony automated extractor is a modular, flexible system that automates the purification of nucleic acids (DNA and RNA) from a wide range of sample types. It offers high-throughput processing capacity and standardized, reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Powerfecal pro kit, by Qiagen (1 mentions)
Onestep pcr inhibitor removal kit, by Zymo Research (1 mentions)
Genexpert system, by Cepheid (1 mentions)
Applied biosystems 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Dsp virus pathogen mini kit, by Qiagen (1 mentions)
Xpert xpress sars cov 2 assay, by Cepheid (1 mentions)
Dsp dna mini kit, by Qiagen (1 mentions)
Blood and tissue kit, by Qiagen (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Qubit dsdna broad range assay kit, by Thermo Fisher Scientific (1 mentions)
Qiamp dna mini kit, by Qiagen (1 mentions)
3 protocols using qiasymphony automated extractor
1
Viral Concentration and Nucleic Acid Extraction
2
Optimized SARS-CoV-2 Detection in Saliva
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All samples were first extracted with the QiaSymphony automated extractor using the DSP virus/pathogen minikit (Qiagen, Germantown, MD) and subsequently tested with a laboratory-developed test (LDT) within 24 h of collection on the Applied Biosystems 7500 fast real-time PCR system (Life Technologies, Carlsbad, CA). Saliva samples that were deemed to have too much mucus by the processing technologist were diluted 1:1 in Tris-EDTA (TE) buffer. Mouth rinse/gargle and saliva samples were also stored in the laboratory at room temperature (21°C) and extracted and tested again on day 1 and day 2 after collection. The previously validated LDT is a triplex reverse transcription-PCR (RT-PCR) assay that targets both the pan-sarbecovirus E gene and the RNA-dependent RNA polymerase (RdRp) gene, as well as the human RNase P gene as a sample positive control, and was previously determined to have a limit of detection of 3.901 log copies/ml (95% confidence interval [CI], 3.048 to 4.755) (5 (link)). A portion of the NPFS and mouth rinse gargle specimens was also tested using the Cepheid Xpert Xpress SARS-CoV-2 assay on the GeneXpert system (Cepheid, Sunnyvale, CA) as per manufacturer instructions.
3
Automated DNA Extraction and Quantification
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Total genomic DNA was extracted using the DSP DNA Mini Kit (Qiagen, Hilden, Germany) and the QIAsymphony automated extractor (Qiagen), or manually either using the QIAmp DNA Mini kit or the Qiagen Blood and Tissue kit (Qiagen). DNA concentration was determined on a Qubit™ fluorometer (ThermoFischer Scientific, Waltham,) using the Qubit™ dsDNA BroadRange assay kit (ThermoFischer Scientific). DNA purity was measured on a Nanodrop 2000 spectrophotometer (ThermoFischer Scientific).
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