dsRNA was extracted either from tissue culture supernatant by BioRobot Universal (Qiagen, UK), or from AHSV infected cells (using Trizol Reagent, Invitrogen, UK) [68] (link). In the first method, 240 μl of infected-tissue-culture supernatant was added to 40 μl of protease (Qiagen, UK) and 360 μl of Lysis Buffer (Roche, UK). Total nucleic acid (50 μl) was extracted from this solution using the BioRobot platform UNIrcV23A V3.0 protocol. Each of the isolates was handled with care to avoid any cross-contamination. Total nucleic acid from uninfected tissue culture supernatants, horse blood or homogenised Culicoides was also extracted using BioRobot Universal (Qiagen, UK).
Universal biorobot
Manufactured by Qiagen
Sourced in United Kingdom
The Universal Biorobot is a versatile automated liquid handling system designed to streamline various laboratory workflows. It features a modular design that can be customized to accommodate a wide range of applications and sample volumes. The system is engineered to deliver precise and accurate liquid handling, contributing to enhanced productivity and reproducibility in the laboratory environment.
Automatically generated - may contain errors
Lab products found in correlation
Qiamp viral rna biorobot kit, by Qiagen (2 mentions)
Mx3000p qpcr system, by Agilent Technologies (2 mentions)
Qiaamp all nucleic acid mdx kit, by Qiagen (2 mentions)
Protease, by Qiagen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Lysis buffer, by Roche (2 mentions)
3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Powerplex y23 system, by Promega (1 mentions)
Schneider s drosophila media, by Thermo Fisher Scientific (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Rneasy 96 universal tissue kit, by Qiagen (1 mentions)
10 protocols using universal biorobot
1
Extraction of dsRNA from AHSV Samples
2
Extracting Double-Stranded RNA from Cell Cultures
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Double‐stranded RNA was extracted either from tissue culture supernatant by BioRobot Universal (Qiagen, UK), or from EHDV‐infected cells (using Trizol Reagent®; Invitrogen, UK) (Attoui et al., 2000 ). In the first method, 240 μl of infected tissue culture supernatant was added to 40 μl of protease (Qiagen) and 360 μl of lysis buffer (Roche, UK). Total nucleic acid (50 μl) was extracted from this solution using the BioRobot platform UNIrcV23A V3.0 protocol. Total nucleic acid from uninfected tissue culture supernatants, cattle blood or homogenized Culicoides was also extracted using BioRobot Universal (Qiagen).
3
Diversity of E. coli Isolates from Malawi
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Sixty blood culture and 26 CSF E. coli isolates were selected from the MLW archives on the basis of diversity of phenotypic resistance profiles and clinical source of isolation. Isolates originated from paediatric (<16 years old) and adult (≥16 years old) patients from the period 1996–2014. Eight rectal swab isolates, all from 2009, were also included. Whole-genome DNA extraction for selected isolates was done at MLW laboratories using the Qiagen Universal Biorobot (Limburg, the Netherlands), following the manufacturer’s instructions.
4
Y-STR Typing using PowerPlex Y23 System
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Y-short tandem repeat (STR) typing was performed using the PowerPlex® Y23 System (Promega). This kit allows for simultaneous analysis of 23 loci on the Y-chromosome.
Automated PCR setup of the extracted DNA was performed on the QIAGEN Universal Biorobot. PowerPlex® Y23 amplification was performed according to the manufacturer’s recommendations. PCR products were separated and detected on an ABI PRISM® 3130xl Genetic Analyzer, using POP-4® Polymer. The results were analyzed with GeneMapper® ID v3.2 software. Allele designation was in accordance with the bins and allelic ladder panels provided within the kit macro. These data have been submitted to the Y-STR Haplotype Reference Database (YHRD) and are now available under the following accession number:YA004186, as well as inS1 Table .
Automated PCR setup of the extracted DNA was performed on the QIAGEN Universal Biorobot. PowerPlex® Y23 amplification was performed according to the manufacturer’s recommendations. PCR products were separated and detected on an ABI PRISM® 3130xl Genetic Analyzer, using POP-4® Polymer. The results were analyzed with GeneMapper® ID v3.2 software. Allele designation was in accordance with the bins and allelic ladder panels provided within the kit macro. These data have been submitted to the Y-STR Haplotype Reference Database (YHRD) and are now available under the following accession number:YA004186, as well as in
5
Influenza Virus Detection and Typing
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RNA was extracted from the nasal swab and tissue suspensions using the QIAmp viral RNA BioRobot kit customised for APHA in conjunction with a Universal BioRobot (Qiagen, Manchester, UK) (Slomka et al. 2009 (link)). Real-time reverse transcription polymerase chain reaction (RRT-PCR) testing of the RNA extracts comprised (1) the Matrix (M)-gene assay for generic IAV detection using the primers and probes of (Nagy et al. 2010 (link)) and (2) H5 and H7 IAV RRT-PCR assays to test for notifiable avian influenza (Slomka et al. 2007 (link), 2009 (link)). For each RRT-PCR assay, samples producing a threshold cycle (CT) value <36.0 were considered positive (Slomka et al. 2010 (link)). The RNA was also tested by an IAV N1-specific RRT-PCR according to the procedure described by Payungporn et al. (2006) (link), an IAV N5-specific RRT-PCR (James et al. 2018 ) and two IAV subtype N8-specific RRT-PCRs (James et al. 2018 ) with the same positive/negative acceptance criteria. All amplifications were carried out in an MX3000P qPCR System (Agilent).
6
Quantifying Schmallenberg Virus in Culicoides
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Total nucleic acid was extracted from the homogenates of Culicoides heads using a Universal Biorobot (Qiagen) with a QIAamp® All Nucleic Acid MDx Kit (Qiagen) following the manufacturer’s recommended instructions. Schmallenberg virus RNA in the resultant extractions was the assessed using a semi-quantitative RT-qPCR targeting the S segment of the genome [1 , 20 (link)]. A Cq cut-off value of < 35 was used to define SBV infection [15 (link)].
7
Quantifying ASFV Genome Copies by qPCR
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Samples were analysed by qPCR to determine the quantity of ASFV genome copies, according to the procedure described in King et al. [26 (link)] with slight modifications. The DNA was extracted with the QIAamp® All Nucleic Acid Kit MDx Kit (Qiagen, UK) using an automated Qiagen Universal BioRobot (Qiagen, UK). After the DNA extraction procedure, the cartridge was processed for the qPCR. The target for amplification of the ASFV genome was the conserved p72 gene segment, using the following primers: 5′-CTG CTC ATG GTA TCA ATC TTA TCG A-3′ and 5′-GAT ACC ACA AGA TC(AG) GCC GT-3′. Analysis was performed using the MxPro software and the qPCR procedure included the following step: denaturation (95 °C), annealing (58 °C) and elongation (72 °C). The quantity of ASFV genome was calculated using the standard curve and expressed as genome copies per millilitre (/mL).
8
Quantifying Schmallenberg Virus in Culex pipiens
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Whole Cx. pipiens, decapitated bodies and heads were homogenized individually for 1 min at 25hz in 100μl of Schneider’s Drosophila Media (Gibco: Life Technologies, Paisley, UK) containing 10% FBS using a TissueLyser (Qiagen, UK) and 3mm stainless steel beads (Dejay Distribution Ltd., UK). Nucleic acid extraction of SBV from samples was subsequently carried out using a Universal Biorobot (Qiagen, UK) in a 96-well format using a QIAamp All Nucleic Acid MDx Kit (Qiagen, UK). Schmallenberg virus RNA in Cx. pipiens samples was quantified using a sqPCR that targeted the S segment of the genome [23 (link)]. Comparisons of RNA quantities were made using Cq values generated from samples.
9
RNA Extraction from Tissue Samples
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Tissue samples were processed using a Qiagen's Universal Biorobot, with the compatible RNA purification kit (RNeasy 96 Universal Tissue Kit), according to the manufacturer's recommendation. Extracted total RNA was eluted in a final volume of 100 µl of the supplied kit elution buffer.
10
Multiplex Detection of Avian Viruses
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Total nucleic acid was extracted from the single oropharyngeal and cloacal swabs, and four standard tissue suspensions (prepared as described for attempted VI) submitted from the statutory NAD investigations using the QIAmp viral RNA BioRobot kit in conjunction with a Universal BioRobot (QIAGEN, United Kingdom [UK]). Nucleic acids were screened by rRT-PCR for (i) generic detection of influenza A virus targeting the matrix (M) gene [12 (link)] and (ii) for specific detection of H5 and H7 AIVs [12 (link), 16 ]. Samples producing a threshold cycle (CT) value <36.0 were considered positive [12 (link), 17 (link)]. All samples were simultaneously screened for APMV-1 using a large polymerase (L) gene-specific rRT-PCR [13 (link)]. A positive result using this assay was denoted by a CT value <37.0. All PCR amplifications were carried out in an MX3000P qPCR System (Agilent).
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