Kingfisher flex system

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, United Kingdom

The KingFisher Flex System is an automated sample preparation instrument designed for high-throughput nucleic acid extraction and purification. It utilizes magnetic particle technology to efficiently and reproducibly process multiple samples simultaneously. The system is compatible with a variety of sample types and can be used in a range of laboratory and research applications.

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Lab products found in correlation

Nucleomag vet kit, by Macherey-Nagel (7 mentions) Tissuelyser 2 tissue homogenizer, by Qiagen (5 mentions) Perfecta qpcr toughmix, by Quanta Biosciences (5 mentions) Mag max viral pathogen kit, by Thermo Fisher Scientific (5 mentions) Magmax cell free dna isolation kit, by Thermo Fisher Scientific (4 mentions) Fragment analyzer, by Agilent Technologies (3 mentions) Fastselect, by Qiagen (3 mentions) Novaseq 6000 s4 flow cell, by Illumina (3 mentions) Quick rna magbead kit, by Zymo Research (3 mentions) Universal plus mrna seq, by Tecan (3 mentions) Nuquant, by Tecan (3 mentions) Magmax mirvana total rna isolation kit, by Thermo Fisher Scientific (3 mentions) Magmax ffpe dna rna ultra kit, by Thermo Fisher Scientific (3 mentions) Magmax viral pathogen nucleic acid isolation kit, by Thermo Fisher Scientific (3 mentions) Miseq platform, by Illumina (3 mentions) Magmax dna multi sample ultra 2.0 kit, by Thermo Fisher Scientific (3 mentions) Qiaamp circulating nucleic acid kit, by Qiagen (2 mentions) Buccal swab dna kit, by Promega (2 mentions) Dnase 1, by Promega (2 mentions) Maxwell rsc instrument, by Promega (2 mentions)

Spelling variants of this product

KingFisher Flex (159 citations) KingFisher Flex Purification System (126 citations) KingFisher Flex extraction system (6 citations) Kingfisher 96 Flex system (6 citations) KingFisher™ Flex automated purification system (5 citations) KingFisher Flex sample purification system (3 citations) KingFisher Flex automated system (2 citations) Kingfisher Flex System instrument (2 citations) KingFisher Flex Magnetic Particle Processing System (2 citations) KingFisher Flex robotic DNA isolation system (2 citations)

88 protocols using kingfisher flex system

PRRSV Serological and Virological Profiling

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Serological (ELISA) and virological (PCR) tests were performed on blood samples from different age groups to determine the time of infection. This process was used to build up a farm-specific eradication protocol, including not only the vaccination regime, but also internal biosecurity measures.
PRRSV ELISA tests from pig sera were carried out applying the INgezim PRRS Universal ELISA Kit (Ingenasa, Madrid, Spain) according to the recommendations of the manufacturer.
RNA from serum samples was extracted with the KingFisher Flex system (ThermoFisher, Waltham, Ma, USA) using MagAttract 96 cador Pathogen Kit. PCR was performed in Rotor-Gene Q (Qiagen) real-time PCR machine using the virotype PRRSV TR-PCR Kit (Qiagen) according to the manufacturer’s instructions.
Samples positive by PCR were subjected to sequencing. The viral ORF5 was sequenced preferably, but in case of failure, the viral ORF7 was also sequenced. Sequencing was performed with the Sanger method using BigDye 3.1 kit (Applied Biosystems, Foster City, CA, USA) on ABI 3500 sequencer (Applied Biosystems). Chromatograms were analysed and edited manually using the BioEdit software version 7.2, available at http://www.mbio.ncsu.edu/BioEdit/bioedit.html [22 ].

Bálint Á., Molnár T., Kecskeméti S., Kulcsár G., Soós T., Szabó P.M., Kaszab E., Fornyos K., Zádori Z., Bányai K, & Szabó I. (2021). Genetic Variability of PRRSV Vaccine Strains Used in the National Eradication Programme, Hungary. Vaccines, 9(8), 849.

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Plasma DNA Extraction and Quantification

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20 ml of blood was centrifuged at 1500 r.p.m. for 10 min within 3 h of collection. Plasma was isolated and frozen at −80 °C. Prior to extraction, plasma samples were further centrifuged at 14 000 r.p.m. for 10 min at 4 °C. DNA was extracted from 5 ml of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) according manufacturer’s instructions. DNA was eluted in 50 μl of AVE buffer and stored at −20 °C. For the validation cohort, circulating DNA was extracted from 2 ml of plasma using the MagMAX Cell-free DNA Isolation kit (ThermoFisher Scientific, Waltham, MS, USA) on a KingFisher Flex System (ThermoFisher Scientific) according to the manufacturer’s instructions. The resulting DNA was eluted in 50 μl of MagMAX Cell-free DNA Elution Solution. Plasma DNA was quantified using a Bio-Rad QX200 ddPCR system- using ribonuclease P as a reference gene as previously described (Garcia-Murillas et al, 2015 ).

Lee J.Y., Garcia-Murillas I., Cutts R.J., De Castro D.G., Grove L., Hurley T., Wang F., Nutting C., Newbold K., Harrington K., Turner N, & Bhide S. (2017). Predicting response to radical (chemo)radiotherapy with circulating HPV DNA in locally advanced head and neck squamous carcinoma. British Journal of Cancer, 117(6), 876-883.

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Bulk RNA-seq for Single-cell Demultiplexing

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Bulk RNA-seq data was generated to extract genotype information, so that single-cells could be demultiplexed and matched to single donors. For each donor, RNA was extracted from PBMCs using the Quick RNA MagBead kit (Zymo Research) on a KingFisher Flex system (Thermo Fisher Scientific) according to the manufacturer’s protocol. RNA integrity was measured with the Fragment Analyzer (Agilent) and library generation was continued when integrity was at least 6. Total RNA-sequencing libraries were depleted from ribosomal and hemoglobin RNAs, and generated using FastSelect (Qiagen) and Universal Plus mRNA-seq with Nu Quant (Tecan) reagents. Pooled libraries were PE100 sequenced on an HiSeq4000 or PE150 sequenced on an Illumina NovaSeq 6000 S4 flow cell at the CZ Biohub.

van der Wijst M.G., Vazquez S.E., Hartoularos G.C., Bastard P., Grant T., Bueno R., Lee D.S., Greenland J.R., Sun Y., Perez R., Ogorodnikov A., Ward A., Mann S.A., Lynch K.L., Yun C., Havlir D.V., Chamie G., Marquez C., Greenhouse B., Lionakis M.S., Norris P.J., Dumont L.J., Kelly K., Zhang P., Zhang Q., Gervais A., Le Voyer T., Whatley A., Si Y., Byrne A., Combes A.J., Rao A.A., Song Y.S., Fragiadakis G.K., Kangelaris K., Calfee C.S., Erle D.J., Hendrickson C., Krummel M.F., Woodruff P.G., Langelier C.R., Casanova J.L., Derisi J.L., Anderson M.S, & Ye C.J. (2021). Type I interferon autoantibodies are associated with systemic immune alterations in patients with COVID-19. Science Translational Medicine, 13(612), eabh2624.

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SARS-CoV-2 RNA Extraction by Maxwell and KingFisher

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RNA extraction was performed using two platforms for parallel sample processing:

By using the Maxwell RSC instrument (Promega AS4500), total RNA was extracted from 300 µL of VTM with the buccal swab DNA kit (Promega AS1640). The following modifications were introduced to extract total RNA as opposed to total nucleic acids: Samples were incubated for 30 min at 65°C for Proteinase K digestion and virus deactivation, and DNase I (Promega) was added to the reagents cartridge to remove genomic DNA during nucleic acids extraction. Total RNA was eluted in 50 µL of nuclease-free water.

By using the KingFisher flex system (Thermo Fisher Scientific), RNA was extracted from heat-inactivated nasopharyngeal swab samples in batches of 96 samples, following the manufacturer's instructions and the MagMax mirVana total RNA isolation kit (Thermo Fisher Scientific A27828). Briefly, 250 µL of nasopharyngeal swab collection was lysed in lysis buffer and β-mercaptoethanol and subsequently bound to magnetic beads and loaded into the KingFisher flex instrument. A DNase I treatment step was performed as part of the instrument protocol, and RNA samples were eluted in 50 µL of elution buffer and immediately stored at −80°C.

Maurano M.T., Ramaswami S., Zappile P., Dimartino D., Boytard L., Ribeiro-dos-Santos A.M., Vulpescu N.A., Westby G., Shen G., Feng X., Hogan M.S., Ragonnet-Cronin M., Geidelberg L., Marier C., Meyn P., Zhang Y., Cadley J., Ordoñez R., Luther R., Huang E., Guzman E., Arguelles-Grande C., Argyropoulos K.V., Black M., Serrano A., Call M.E., Kim M.J., Belovarac B., Gindin T., Lytle A., Pinnell J., Vougiouklakis T., Chen J., Lin L.H., Rapkiewicz A., Raabe V., Samanovic M.I., Jour G., Osman I., Aguero-Rosenfeld M., Mulligan M.J., Volz E.M., Cotzia P., Snuderl M, & Heguy A. (2020). Sequencing identifies multiple early introductions of SARS-CoV-2 to the New York City region. Genome Research, 30(12), 1781-1788.

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Wastewater Viral RNA Extraction

Cited 2 times

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As described above, only wastewater samples from the FD manholes were tested daily, following the two-tier group sampling strategy. All wastewater samples were first heat-inactivated in a water bath (60 ^oC for 30 min) prior to laboratory analysis to ensure the safety of laboratory personnel [47] (link). The effectiveness of thermal treatment for virus inactivation was reported for SARS-CoV-2 without affecting RNA integrity [49] (link), [48] (link). After heat inactivation, samples were then subjected to viral concentration. Briefly, an aliquot of 20 mL of heat-inactivated raw sewage was centrifuged at 4000 g for 30 min at 4 ^oC to remove larger solid particles/debris and bacteria. 15 mL of this supernatant was then concentrated using centrifugal ultrafiltration at 4000 g for 20 min at 4 ^oC (Amicon® Ultra-15, Merck) to produce virus concentrates. Viral RNA was extracted from 200 µl of the concentrated sample using the KingFisher Flex System and MagMAX Viral Pathogen II Kit (ThermoFisher, USA), according to manufacturer's instructions.

Mohapatra S., Bhatia S., Senaratna K.Y., Jong M.C., Lim C.M., Gangesh G.R., Lee J.X., Giek G.S., Cheung C., Yutao L., Luhua Y., Yong N.H., Peng L.C., Wong J.C., Ching N.L, & Gin K.Y. (2022). Wastewater surveillance of SARS-CoV-2 and chemical markers in campus dormitories in an evolving COVID − 19 pandemic. Journal of Hazardous Materials, 446, 130690.

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Capripox Virus Genome Detection

Cited 3 times

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Organ samples were homogenized in a serum-free medium using the TissueLyser II tissue homogenizer (Qiagen, Hilden, Germany). Genome extraction of samples taken during the animal trial and homogenized tissue samples was performed utilizing the KingFisher Flex System (Thermo Scientific, Darmstadt, Germany) using the NucleoMag Vet kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions with volume modifications as described previously [46 (link)]. To control successful DNA extraction and inhibition-free amplification, an internal control DNA (IC2-DNA) was added to the samples during the extraction process [47 (link),48 (link)]. Analyses of the presence of the capripox virus genome were performed using the pan capripox real-time qPCR of Bowden et al. [49 (link)] with a modified probe [50 (link)], utilizing the PerfeCTa qPCR ToughMix (Quanta BioSciences, Gaithersburg, MD, USA) on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA).

Wolff J., Tuppurainen E., Adedeji A., Meseko C., Asala O., Adole J., Atai R., Dogonyaro B., Globig A., Hoffmann D., Beer M, & Hoffmann B. (2021). Characterization of a Nigerian Lumpy Skin Disease Virus Isolate after Experimental Infection of Cattle. Pathogens, 11(1), 16.

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Homogenization and Viral Genome Analysis

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For the homogenization of organ samples in a serum-free medium, the TissueLyzer II tissue homogenizer (Qiagen, Hilden, Germany) was used. Subsequently, the DNA of all samples taken during the study was extracted using the KingFisher Flex System (Thermo Scientific, Darmstadt, Germany) and the NucleoMag Vet kit (Macherey-Nage, Düren, Germany) according to the manufacturer’s instructions. During DNA extraction, an internal control-DNA (IC-2 DNA) was added to the samples to control successful DNA extraction and inhibition-free amplification [44 (link),45 (link)]. The analysis of the viral genome load in the samples was performed using the PerfeCTa qPCR ToughMix (Quanta BioSciences, Gaithersburg, MD, USA) and the already described pan capripox real-time qPCR of Bowden et al. [6 (link)] with a modified probe [46 (link)].

Wolff J., Beer M, & Hoffmann B. (2023). Cross-Protection of an Inactivated and a Live-Attenuated Lumpy Skin Disease Virus Vaccine against Sheeppox Virus Infections in Sheep. Vaccines, 11(4), 763.

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Automated Protein Purification and Preparation

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For each conditioned media sample, the volume corresponding to 20 µg of protein was taken and supplemented with 20% sodium dodecyl sulfate (SDS) to a final concentration of 4%. The samples were boiled at 95 °C for 10 min while shaking at 800 rpm on a Thermoshaker (Eppendorf). The samples were reduced with 5 mM dithiothreitol for 30 min at room temperature followed by alkylation with 15 mM iodoacetamide at 50 °C for 30 min in the dark. Samples were processed using the single-pot solid-phase enhanced sample preparation (SP3) [10 (link),11 (link)]. In short, protein purification, digestion and peptide clean-up were performed using a KingFisher Flex System (Thermo Fisher Scientific) and carboxylate-modified magnetic particles (GE Life Sciences; GE65152105050250, GE45152105050250) after the manufacturer’s instructions. Resulting digest solution and water elution were combined, dried and re-solubilized in 15 µL of MS sample buffer (3% acetonitrile, 0.1% formic acid).

Car I., Dittmann A., Klobučar M., Grbčić P., Kraljević Pavelić S, & Sedić M. (2023). Secretome Screening of BRAFV600E-Mutated Colon Cancer Cells Resistant to Vemurafenib. Biology, 12(4), 608.

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Viral Genome Extraction and Quantification

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Due to the limited sample volume and the decision to use 1 mL for the passaging of samples in cell culture, 10 µl of each sample was diluted in 90 µl of the respective medium (PBS, ZB, or FCS) to receive 100 µl for the extraction of DNA. Nucleic acid extraction was done using the NucleoMag Vet kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions on the KingFisher Flex System (Thermo Scientific, Darmstadt, Germany), with some modifications in the volume of certain substances [22 (link)]. For the four CaPV isolates, the viral genome loads were determined using a pan-capripox real-time qPCR amplifying a region in the p-32 gene [2 (link)] in combination with a modified probe [23 (link)]. PCPV genomes were detected using a real-time qPCR assay targeting the B2L gene [24 (link)]. An internal control DNA (IC2-DNA) was added for the control of successful DNA extraction [25 (link)]. For both used real-time qPCR assays, analytic sensitivity of less than 10 copy numbers per PCR reaction are reported [2 (link),24 (link)].

Wolff J., Beer M, & Hoffmann B. (2020). Thermal Inactivation of Different Capripox Virus Isolates. Microorganisms, 8(12), 2053.

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Evaluating Anti-CHIKV Antibody Efficacy

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Vero cells (2 x 10⁵ cells/ml) were inoculated with CHIKV 181/25 (MOI 0.01) at 37°C for 4 h. Infected cells were rinsed with PBS and incubated with anti-CHIKV mAbs (10 μg/ml), mycophenolic acid (50 μg/ml, Sigma), or a mock control. Infected cells were harvested at 2, 6, or 10 h post addition of mAbs or mycophenolic acid, and intracellular viral RNA was extracted using the KingFisher Flex system (Thermo Fisher). CHIKV RNA was quantitated as described above in “Egress inhibition assays”.

Kim A.S., Kafai N.M., Winkler E.S., Gilliland TC J.r., Cottle E.L., Earnest J.T., Jethva P.N., Kaplonek P., Shah A.P., Fong R.H., Davidson E., Malonis R.J., Quiroz J.A., Williamson L.E., Vang L., Mack M., Crowe JE J.r., Doranz B.J., Lai J.R., Alter G., Gross M.L., Klimstra W.B., Fremont D.H, & Diamond M.S. (2021). Pan-protective anti-alphavirus human antibodies target a conserved E1 protein epitope. Cell, 184(17), 4414-4429.e19.

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