HIV-1 RNA was extracted from plasma by automated magnetic silica extraction using the EasyMAG extractor (bioMérieux). The extracted RNA was amplified by RT–PCR and nested PCR to obtain the HIV-1 pol region, using primers designed by WHO for protease (PR) and reverse transcriptase (RT) amplification and ANRS primers for integrase (IN) amplification,25 ,26 as previously described.27 (link),28 (link) PCR amplicons were purified with illustraTM ExoProStarTM (Cytiva) and sequenced by Macrogen Inc. Lasergene software was used to assemble and manually edit the sequences. Viral sequences included the complete HIV-1 PR (codons 1–99), partial RT (1–345) and IN (48–285) for genotyping study of DRMs to PIs, NRTIs, NNRTIs and integrase strand transfer inhibitors (INSTIs). Stanford HIVdb Program v9.0 (Stanford University, Palo Alto, CA, USA) (https://hivdb.stanford.edu/hivdb/by-sequences/ ) was used to characterize DRMs in pre-treated children/adolescents and predict the resistance level to 25 ARVs in pol genotypes. Transmitted drug resistance (TDR) mutation prevalence was established among the ART-naive population by the WHO TDR list 2009 implemented in the Calibrated Population Resistance (CPR) tool v8.0 (https://hivdb.stanford.edu/cpr/ ) and by the Stanford algorithm v9.0, both available on the Stanford HIV website (https://hivdb.stanford.edu/ ).7 (link)
Easymag extractor
Manufactured by bioMérieux
Sourced in France
The EasyMAG extractor is a fully automated nucleic acid extraction instrument designed for use in clinical and research laboratories. It utilizes a magnetic bead-based technology to efficiently isolate high-quality nucleic acids (DNA and RNA) from a variety of sample types. The EasyMAG extractor provides a standardized and reproducible extraction process, ensuring consistent results.
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Lab products found in correlation
Ttv r gene kit, by bioMérieux (3 mentions)
Exoprostar, by Cytiva (1 mentions)
Peg 6000, by Merck Group (1 mentions)
Nuclisens kit, by bioMérieux (1 mentions)
Rotor gene q system, by Qiagen (1 mentions)
Nuclisens easymag kit, by bioMérieux (1 mentions)
7300 rt pcr system, by Thermo Fisher Scientific (1 mentions)
Geneamp pcr system 9700 thermocycler, by Thermo Fisher Scientific (1 mentions)
Ttv r gene assay, by bioMérieux (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
9 protocols using easymag extractor
1
HIV-1 Genotypic Resistance Profiling
2
Sewage Virus Concentration and Extraction
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The 12 samples were concentrated from 40 ml raw sewage by adding 10 ml of 50% polyethylene glycol (PEG 6,000; Sigma-Aldrich, St-Quentin France; Sima et al., 2011 (link)). After incubation and centrifugation, the PEG pellet was suspended in 1 ml of deionized distilled water (DDW) with a vortex mixer, and nucleic acids were extracted with an automatic easyMAG extractor (bioMérieux, Lyon, France) and the NucliSENS kit (bioMérieux). Norovirus detection was performed as described below.
3
Comprehensive Fecal Pathogen Screening
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Fecal intestinal pathogens (n = 15) were assessed by polymerase chain reaction at the Hospital for Sick Children, Toronto, Canada, with the use of the Gastrointestinal Pathogen Panel (Luminex Molecular Diagnostics) according to the manufacturer’s instructions. In brief, nucleic acids were extracted from 100–150 mg of formed or 100 μL of loose stool by easyMAG extractor (bioMerieux) and underwent multiplexed polymerase chain reaction for Salmonella spp., Shigella spp., Campylobacter jejuni/coli, Yersiniaenterocolitica (pathogenic serotype only), Escherichia coli 0157:H7, non-0157 shiga-like toxin-producing E. coli, Clostridium difficile toxin A/B, enterotoxigenic E. coli, Vibrio cholerae, rotavirus A, adenovirus 40/41, norovirus GI/II, Giardia lamblia, Entamoeba histolytica, and Cryptosporidium parvum.
4
Quantification of TTV DNA in Plasma
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Total nucleic acids (elution volume 50μL) were extracted from 200μL of plasma sample using an easyMag extractor (bioMérieux, Marcy-l’Etoile, France) following the manufacturer’s instructions. The presence and viral load of TTV DNA were then determined using the TTV R-GENE® kit (available for research use only, not for diagnostic procedure; Ref#69–030, bioMérieux) as previously described [37 (link)]. Real-time PCR amplification was performed according to manufacturer’s instructions on a Stratagene® Mx3005P™ platform (Stratagene, La Jolla, CA, USA).
5
Comprehensive Respiratory Virus Screening
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Samples were screened for HADV, HBOV, HCOV, HMPV, HPIV, HRSV, and IV nucleic acids with real-time (RT)-PCR or reverse transcription (rt)-RT-PCR assays. Nucleic acid extraction was performed with the NucliSENS® easyMAG® kit on the EasyMAG extractor (BioMérieux, Italy) and rt with the RT-kit plus (ELITechGroup Molecular Diagnostics, France) on the GeneAmp PCR System 9700 thermocycler (Applied Biosystems, Thermo Fischer Scientific-Waltham, USA). Extracted nucleic acids were tested with Influenza A/B Q-PCR Alert AmpliMIX kit (ELITechGroup) on the 7300 RT-PCR system (Applied Biosystems). The Respiratory Multi Well System (MWS) r-gene™ assay (Argene/BioMérieux) was used to detect ADV/HBOV, HCOV/HPIV, and RSV/HMPV on the Rotor-Gene Q system (Qiagen, Germany). Assays were performed according to the manufacturer's instructions.
6
Quantification of Torque Teno Virus
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TTV viral DNA (elution volume 50 μL) was extracted from 200 μL of plasma sample using an easyMag extractor (bioMérieux, Marcy-l’Etoile, France) following the manufacturer’s instructions. The presence and viral load of TTV were then determined using the TTV R-GENE® kit (available for research use only, not for diagnostic, Ref#69-030; bioMérieux, Marcy-l’Etoile, France) as previously described [19 (link),20 (link)]. Log of TTV DNA copy number/mL (Log copies/mL) plasma are used to describe TTV viral load. The lowest viral load detected was 0.46 Log copies/mL.
7
Quantification of TTV-DNA Viral Load
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Quantification of TTV-DNA viral load from whole blood was performed using the standardized TTV R-GENE® kit (bioMérieux, France), as specified by the manufacturer (40 (link)). Viral DNA was extracted from 200 μl EDTA whole blood using an easyMAG extractor (bioMérieux, France) with 140 μl of silica and a 50 μl elution volume. Ten μl of eluate was added to 15 μl of ready-to-use amplification premix (TTV R-GENE® assay, bioMérieux, France). In each run, an internal control and four quantification standards provided by the manufacturer were included. Amplification was performed according to manufacturer’s instructions on CFX96™ Real-Time PCR Detection System (Bio-rad Hercules, CA, USA). The limit of detection was 2.4 log10 copies/ml and the quantification range was between 2.4 to 9 log10 copies/ml.
8
Antimicrobial Susceptibility Testing and DNA Extraction
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Antimicrobial susceptibility testing was carried out by the disc diffusion method using Mueller-Hinton agar plates supplemented with lysed horse blood (5%) and β-NAD DNA extraction was performed using the automated easyMAG® extractor (bioMérieux, France) according to the protocol suggested by the manufacturer.
Extracted bacterial DNA was eluted with 25 μl elution buffer and stored at -20°C.
Extracted bacterial DNA was eluted with 25 μl elution buffer and stored at -20°C.
9
Maternal CMV Infection Detection
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Type of maternal infection and dating of a maternal primary infection were based on serological results as described elsewhere (3, 5) . Saliva samples were collected with flocked swabs and then discharged into transport medium (Copan, ESWABR1, Labelians, Nemours, France). DNA was extracted from 200 L of saliva or of whole blood sample on the EasyMag Extractor (BioMérieux, Marcy l'Etoile, France) and amplified using the real time PCR CMV R Gene assay (Argène BioMerieux). The limit of detection of the method is 2.5 log10 copies/mL in saliva and 2.6 IU/mL in whole blood; the limit of quantification is 2.7 log10 copies/mL in saliva and 2.8 IU/mL in whole blood.
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