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17 protocols using qiaamp minielute virus spin kit

1

Microbiome profiling from frozen nasopharyngeal samples

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The nasopharyngeal samples frozen at –80 °C were used for DNA isolation with the QIAamp MiniElute Virus Spin Kit (Quiagen, Hilden, Germany), following the protocol recommended by the manufacturer. The DNA obtained was quantified with a Qubit 4 Fluorometer, using a Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, Massachusetts, United States). The microbiota amplicon sequencing was performed following the protocol of the 16S Metagenomics Sequencing Library Preparation recommended by Illumina. The V3 and V4 region from 16S rRNA gene were amplified by PCR, and then the fragments obtained were sequenced in the MiSeq system with V3 reagents (600 cycle, 2 × 300 bp).
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2

Microbiome DNA Sequencing Protocol

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The nasopharyngeal samples frozen at -80 °C were used for DNA isolation with the QIAamp MiniElute Virus Spin Kit (Quiagen, Hilden, Germany), following the protocol recommended by the manufacturer. The DNA obtained was quantified with a Qubit 4 Fluorometer, using a Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, Massachusetts, United States). The microbiota amplicon sequencing was performed following the preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
protocol of the 16S Metagenomics Sequencing Library Preparation recommended by Illumina. The V3 and V4 region from 16S rRNA gene were amplified by PCR, and then the fragments obtained were sequenced in the MiSeq system with V3 reagents (600 cycle, 2x300bp).
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3

Molecular Identification of Cutaneous BPV Types

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Molecular identification of BPV-1, -2, -4, -5, and -10 in head and neck skin wart specimens was made with PCR. Oligonucleotide primers were synthesized for the amplification of the L1 gene of BPV-1 and -2, the E7 gene of BPV-4, and the E2 gene for BPV-5 and -10 according to protocols described elsewhere [33 ,34 (link)]. Primers were synthesized by Metabion International AG, Planegg, Germany and used at a 10-µM concentration. DNA extraction was carried out using a QIAamp® MiniElute® Virus Spin Kit (QIAGEN, GmbH, Hilden, Germany) based on the manufacturer’s instructions. Negative control skin samples were also involved. PCR amplification was done following a protocol described elsewhere [33 ], using a Thermo Scientific PCR Master Mix (Thermo Scientific, Waltham, MA, USA). The PCR conditions and time-temperature program was as follows: 95 °C for 10 min for initial melting, 30 cycles of 94 °C for 45 s (melting), 50 °C for 45 s (annealing), and 72 °C for 1 min (extension), followed by 72 °C for 7 min (final extension). Visualization of the PCR products by gel electrophoresis was performed as reported elsewhere [35 ].
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4

HBV Core Gene Amplification and Sequencing

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The genomic region covering the HBV core gene was amplified and sequenced from patients enrolled in this study. Viral DNA was extracted with the QIAamp MiniElute Virus Spin Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. A 700-bp core fragment was amplified in a two-step nested polymerase chain reaction (PCR) using HBV-specific primers HBV core-forward (tgtcaacgaccgaccttgagg), HBV core-reverse (tgtagctcttgttcccaa), HBV core internal-forward (aggctgtaggcataaattggt), and HBV core internal-reverse (ttcccaccttatgagtccaag), as previously described (Supplementary Table 1) (27 (link)). PCR products were directly sequenced and aligned by Cosmogenetech (Seoul, Republic of Korea).
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5

Quantitative CMV Viral Load Assay

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Sample processing was done within 4 h of venepuncture, and plasma was stored continuously at −80°C until use. Viral nucleic acids were extracted from 200 μl of plasma using the QIAamp MiniElute Virus Spin Kit (Qiagen, Limburg, Netherlands). CMV viral load was determined using the RealStar CMV PCR Kit 1.0 (Altona Diagnostics, Hamburg, Germany) following the manufacturer's instructions. Real-time quantitative PCR was done using the ABI Prism 7500 SDS instrument (Applied Biosystems, Waltham, Massachusetts, USA). A single 96-well plate run was used for all HEU and HUU samples, a negative control and four standards calibrated against the first WHO International Standard for Human CMV Nucleic Acid Amplification (these served to generate a standard curve, as quality controls, and as a positive control). The assay limit of detection is estimated to be 92.1 IU/ml. Viraemia was defined as a measurable viral load, as per manufacturer's recommendations, and viral loads are presented as log10 IU/ml of peripheral blood.
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6

Quantitative SHIV RNA Detection in Plasma

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SHIV copy number in plasma was determined by TAQMAN quantitative real-time PCR assay. Viral RNA was extracted from 200 μL of plasma using QIAamp MiniElute Virus Spin Kit (57704, Qiagen) and amplified using the following primer/probe set: SIV Fwd, GTCTGCGTCATCTGGTGCATTC; SIV Rev, CACTAGGTGTCTCTGCACTATCTGTTTTG; probe, 6FAM-CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG-TAMRA. All samples were amplified in triplicate in an Applied Biosystems 7500 Sequence detector using the following program: 48°C for 30 min, 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Values above 50 copies/mL limit of detection were extrapolated from a standard curve.
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7

Detection of Respiratory Viruses by RT-PCR

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Clinical specimens consisted of a nasopharyngeal aspirate (NPA) taken from each patient at admission. All clinical specimens were sent for virological investigation to the Respiratory Virus and Influenza Unit at the National Microbiology Center (ISCIII, Madrid, Spain), NPAs were processed within 24 hours after collection. Upon receipt, three aliquots were prepared and stored at -70°C.
RNA and DNA from 200 μl-aliquots of NPA were extracted by using the QIAamp Mini Elute Virus spin kit in an automated extractor (QIAcube, Qiagen, Valencia, CA). From 2005 to 2010, three conventional multiplex RT-nested-PCR assays were performed to detect a total of sixteen respiratory viruses [9 (link),10 (link),11 (link)]. From 2011 to 2014, detection of HMPV and the other respiratory virus were performed by real time multiplex RT-PCR assays, not published yet, but based on the same equivalent conventional methods (9, 10, 11).
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8

Serological and Molecular Detection of HEV

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Serum was obtained by centrifugation for 10 min at 400 × g and stored at −80°C until required for analysis. Samples were tested for anti‐HEV IgG and anti‐HEV IgM antibodies by commercial ELISA (recomWell HEV IgG/IgM®; Mikrogen Diagnostik, Neuried, Germany) using an automated procedure (Automatic ELISA workstation DS2®, Dynex Technologies). The analyses were carried out in accordance with the instructions provided by the manufacturer using a cut‐off value ≥ 24 U/ml for positive samples. Antibodies quantitative value are presented in U/ml, not related with the WHO international standard. The specimens with a value‐to‐cut‐off ratio between 20 and 24 U/ml were considered borderline. Confirmatory testing was performed using immunoblotting (recomLine HEV IgG/IgM®; Mikrogen Diagnostik, Neuried, Germany), following a manual procedure according to the manufacturer's instructions for all positive samples. RNA was extracted from 400 μl of serum using the commercial QIAamp Mini Elute Virus Spin Kit (QIAgen, Hilden, Germany) by an automated procedure (QIAcube. QIAgen, Hilden, Germany). The purified RNA was eluted in a volume of 50 μl. RT q‐PCR for HEV was performed using the QIAgen One‐Step PCR Kit (QIAgen, Hilden, Germany), following an in‐house protocol described previously by our group (Frías et al., 2021 (link)).
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9

Detecting Enteric Viruses in Stool Samples

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Stool samples were collected in sterile tubes, transferred to the laboratory, and stored at −70°C until further processing. Stool samples were diluted to 10% (w/v) with phosphate-buffered saline and centrifuged. Viral double stranded RNA and DNA were extracted from the supernatant using the QIAamp MiniElute Virus Spin Kit (Qiagen, Hilden, Germany). PCR was performed to detect adenovirus (15 (link)) and reverse transcription PCR (RT-PCR) was performed to detect rotavirus (8 (link)), norovirus (16 (link)), and astrovirus (17 (link)) as described previously (18 ). Multiplex PCR using a Seeplex Diarrhea-V ACE detection kit (Seegene Inc., Seoul, Korea) was also performed to detect astrovirus, group A rotavirus, enteric adenovirus, and norovirus, according to the manufacturer's instructions (19 (link)). Amplification products were examined by electrophoresis on 1.5% agarose gels and documented with the Bio-Rad Gel Doc 1000 Documentation System (BioRad, Hercules, CA, USA). The samples were scored as positive for rotavirus if the rotavirus antigen test and/or PCR reactions were positive. The samples were scored as positive for norovirus, adenovirus, or astrovirus if the PCR reactions were positive.
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10

Viral RNA/DNA Extraction and Reverse Transcription

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Viral RNA and DNA were extracted from 200 μl nasopharyngeal swab using a QIAamp MiniElute Virus Spin Kit (Qiagen, Germany) according to the manufacturer’s instructions. Reverse transcription of viral RNA was performed using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA).
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