Flex purification system

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The Flex Purification System is a laboratory equipment designed for sample purification. It utilizes a flexible modular approach to accommodate various purification techniques and sample types.

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12 protocols using flex purification system

SARS-CoV-2 Detection in Saliva Samples

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The presence of SARS-CoV-2 was confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). 200 μL of VTM was used for RNA purification. RNA was extracted from the clinical samples on Kingfisher flex purification system Thermo Fisher using MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (thermos fisher). Reactions were performed in 20 μL final volume reaction containing 5 μL of extracted RNA, rRT-PCR was performed using CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and Bosphore Novel Coronavirus (2019-nCoV) PCR Detection Kit v4 (Anatolia, Turkey), which targeted the RdRP, N and E genes of SARS-CoV-2. In this assay, a RNase P gene region is used as an endogenous internal control for the analysis of biological samples. It is normally used to ensure the quality of the test, at extraction and PCR levels and to exclude the false negative results.
Thus, in order to evaluate possible variability in the amount of material retrieved from saliva specimen before and after mouth wash we utilized RNase P as reference gene to normalize the input data.
To compare the paired samples before and after mouth wash, we calculated a Ct value modified according to the ratio of sample RNase P and mean RNase P Ct values.³¹ (link)

\frac{sample SarsCoV 2 Ct value x sample RNaseP Ct}{mean RNaseP Ct value}

Elzein R., Abdel-Sater F., Fakhreddine S., Hanna P.A., Feghali R., Hamad H, & Ayoub F. (2021). In vivo evaluation of the virucidal efficacy of chlorhexidine and povidone-iodine mouthwashes against salivary SARS-CoV-2. A randomized-controlled clinical trial. The Journal of Evidence-Based Dental Practice, 21(3), 101584.

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Automated Nucleic Acid Extraction from Patient Samples

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Nucleic acids were isolated using the MagMAX-96 Total RNA Isolation Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA; Cat. No. AM1830). Briefly, 200 µL PBS were taken from patient swab sample and mixed with 265 µL binding buffer, 5 µL proteinase K (20 mg/mL) and 5 µL extraction control (Thermo Fisher Scientific, Waltham, Massachusetts, USA; Cat. No. AM1830) according to the KingFisher extraction protocol for 200 µL sample volume (Thermo Fisher Scientific, Waltham, Massachusetts, USA). After incubation at room temperature for at least 15 min, samples were transferred from tubes into 96-well KingFisher deep well plates (Thermo Fisher Scientific, Waltham, MA, USA) containing 280 µL isopropanol and 2 µL Mag-Bind particles per well, using a KingFisher Flex purification system (Cat. No. 5400620).

Sonnleitner S.T., Prelog M., Sonnleitner S., Hinterbichler E., Halbfurter H., Kopecky D.B., Almanzar G., Koblmüller S., Sturmbauer C., Feist L., Horres R., Posch W, & Walder G. (2022). Cumulative SARS-CoV-2 mutations and corresponding changes in immunity in an immunocompromised patient indicate viral evolution within the host. Nature Communications, 13, 2560.

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Ectoparasites Identification and Bartonella Detection

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We collected ectoparasites from the skin and pelage of bats and stored them in microcentrifuge tubes with 70% ethanol. Ectoparasite species were identified by using available morphologic keys (17 (link)), and identifications were later confirmed by sequencing of the mitochondrial 16S rRNA and cytochrome oxidase I (COI) genes (18 ,19 (link)).
Using a Bullet Blender Gold homogenizer (Next Advance, Averill Park, NY, USA), we homogenized whole ectoparasites in Navy Eppendorf bead tubes (Next Advance) containing 400 μL brain–heart infusion broth (CDC, Atlanta, GA, USA). We extracted DNA from the homogenates by using the KingFisher Flex Purification System and the associated MagMAX Pathogen RNA/DNA Kit (both ThermoFisher, Waltham, MA, USA) according to the manufacturer’s protocols. Detection of Bartonella DNA in ectoparasite samples was performed by nested PCR for gltA (20 (link)) because of low concentrations of DNA and by conventional PCR for ITS (21 (link)), followed by sequencing and sequence analysis of amplicons.

Bai Y., Osinubi M.O., Osikowicz L., McKee C., Vora N.M., Rizzo M.R., Recuenco S., Davis L., Niezgoda M., Ehimiyein A.M., Kia G.S., Oyemakinde A., Adeniyi O.S., Gbadegesin Y.H., Saliman O.A., Ogunniyi A., Ogunkoya A.B, & Kosoy M.Y. (2018). Human Exposure to Novel Bartonella Species from Contact with Fruit Bats. Emerging Infectious Diseases, 24(12), 2317-2323.

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RNA Extraction Using KingFisher Flex

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Inputs of 200 µL were mixed with 200 µL NAxtra LYSIS BUFER (Lybe Scientific). To each sample, it was added a 600 µL mixture of 20 µL NAxtra MAGNETIC BEADS (Lybe Scientific) in 580 µL isopropanol. Lysis, binding, washing, and elution were performed on the KingFisher Flex Purification System with a 96 Deep-Well Head (Thermo Scientific). Elution was generally performed in 50 µL of nuclease free water. When comparing to the MagMAX viral/pathogen II (MVP II) Nucleic Acid Isolation Kit (Applied Biosystems), elution volumes were 100 µL (Fig. 1).

Ravlo E., Sousa M.M., Andersen L.L., Holberg-Petersen M., Klundby I., Aas P.A., Hagen L., Erlandsen S.E., Malmring J.F., Ali Z., Sharma A., Ottesen V., Bandyopadhyay S, & Bjørås M. (2023). A fast, low-cost, robust and high-throughput method for viral nucleic acid isolation based on NAxtra magnetic nanoparticles. Scientific Reports, 13, 11714.

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Quantitative BTV RNA Extraction and Analysis

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BTV RNA was extracted from 100 μL of tissue culture supernatant or midge homogenate using the KingFisher™ Flex Purification System and the MagVet™ Universal Nucleic Acid Extraction Kit (ThermoFisher Scientific, Paisley, UK) as described previously [47 (link)]. Briefly, 5 μL of sample RNA was analyzed using a BTV-Seg-10-specific RT-qPCR assay [43 (link)] adapted to fast cycling conditions [47 (link)]. To quantify the level of viral RNA, a log-dilution series of the BTV Seg-10 ssRNA transcript (1 × 10⁸–1 × 10¹ copies per µL) was included in duplicate on each plate as a standard. The number of BTV genome copies in a sample was determined by comparison of the C_T values obtained to the standard curve.

Ropiak H.M., King S., Busquets M.G., Newbrook K., Pullinger G.D., Brown H., Flannery J., Gubbins S., Batten C., Rajko-Nenow P, & Darpel K.E. (2021). Identification of a BTV-Strain-Specific Single Gene That Increases Culicoides Vector Infection Rate. Viruses, 13(9), 1781.

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SARS-CoV-2 Amplicon Sequencing Protocol

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NP swab samples were prepared using 100 uL of the primary sample in UTM or VTM mixed with 100uL DNA/RNA shield (Zymo Research, # R1100-250). The 1:1 sample mixture was then extracted using the Omega BioTek MagBind Viral DNA/RNA Kit (Omega Biotek, # M6246-03) on KingFisherTM Flex Purification System with a 96 deep-well head (ThermoFisher, 5400630). Extracted RNA was reverse transcribed to complementary DNA and tiling multiplexed amplicon PCR was performed using SARS-CoV-2 primers Version 3 according to a published protocol (Quick et al., 2017 (link)). Amplicons were ligated with adapters and incorporated with barcodes using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, # E7645L). Libraries were barcoded using NEBNext Multiplex Oligos for Illumina (96 unique dual-index primer pairs) (New England Biolabs, # E6440L) and purified with AMPure XP (Beckman-Coulter, #. Amplicon libraries were then sequenced on either Illumina MiSeq or Novaseq 6000 as 2x150 paired-end reads (300 cycles).

Deng X., Garcia-Knight M.A., Khalid M.M., Servellita V., Wang C., Morris M.K., Sotomayor-González A., Glasner D.R., Reyes K.R., Gliwa A.S., Reddy N.P., Martin C.S., Federman S., Cheng J., Balcerek J., Taylor J., Streithorst J.A., Miller S., Kumar G.R., Sreekumar B., Chen P.Y., Schulze-Gahmen U., Taha T.Y., Hayashi J., Simoneau C.R., McMahon S., Lidsky P.V., Xiao Y., Hemarajata P., Green N.M., Espinosa A., Kath C., Haw M., Bell J., Hacker J.K., Hanson C., Wadford D.A., Anaya C., Ferguson D., Lareau L.F., Frankino P.A., Shivram H., Wyman S.K., Ott M., Andino R, & Chiu C.Y. (2021). Transmission, infectivity, and antibody neutralization of an emerging SARS-CoV-2 variant in California carrying a L452R spike protein mutation. medRxiv.

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Automated Viral RNA Extraction

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Viral RNA had been extracted from nasopharyngeal swab samples using an automated nucleic acid purification system (Thermo Scientific KingFisher Flex Purification System, Waltham, MA, USA) following the MVP_2Wash_200_Flex protocol as recommended by the manufacturer. After extraction, eluted RNA is stored at −20 °C until further processing.

Papa Mze N., Beye M., Kacel I., Tola R., Basco L., Bogreau H., Colson P, & Fournier P.E. (2022). Simultaneous SARS-CoV-2 Genome Sequencing of 384 Samples on an Illumina MiSeq Instrument through Protocol Optimization. Genes, 13(9), 1648.

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Saliva-based SARS-CoV-2 RT-PCR detection

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RNA was extracted from 200 µL of neat saliva as previously described [33] . Briefly, saliva was mixed with a binding solution (265 µL) using the Mag-MAX Viral/Pathogen Nucleic Acid Isolation Kit on a KingFisher Flex Purification system (Thermofisher, Loughborough, UK). The nucleic acid eluates were stored at -20 °C prior to RT-PCR analysis.
RT-PCR analysis cDNA was produced by reverse transcribing the purified viral RNA using a RealStar ® SARS-CoV-2 RT-PCR kit (Altona Diagnostics, Hamburg, Germany). The SARS-CoV-2 E gene sequence region was targeted, using 10 µL of nucleic acid eluate. All Ct values were reported below Ct 40.

Lane D., Allsopp R., Holmes C.W., Slingsby O.C., Jukes-Jones R., Bird P., Anderson N.L., Razavi M., Yip R., Pearson T.W., Pope M., Khunti K., Doykov I., Hällqvist J., Mills K., Skipp P., Carling R., Ng L., Shaw J., Gupta P, & Jones D.J.L. (2024). A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test. Clinical chemistry and laboratory medicine, 62(6).

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Automated RNA Extraction Methods

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Throughout the pandemic the HCPHL used a variety of extraction platforms. RNA extractions were initially performed manually using the QIAamp Viral RNA mini kit (Qiagen) following manufacturer instructions. To improve throughput, automated RNA extraction was implemented on the MagNAPure Compact (Roche), eluting the extracted sample in 100 µls of elution buffer. Additionally, some extractions were either performed on the Qiagen EZ1 Advanced, eluting samples in 120 µls of elution buffer, or on the KingFisher™ Flex Purification System and the MagMAX™ Viral/Pathogen II Nucleic Acid Isolation Kit (ThermoFisher), which eluted the sample in 100 µls of elution buffer.

Stoddard G., Black A., Ayscue P., Lu D., Kamm J., Bhatt K., Chan L., Kistler A.L., Batson J., Detweiler A., Tan M., Neff N., DeRisi J.L, & Corrigan J. (2022). Using genomic epidemiology of SARS-CoV-2 to support contact tracing and public health surveillance in rural Humboldt County, California. BMC Public Health, 22, 456.

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Whole-Genome Sequencing of SARS-CoV-2

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For whole-genome sequencing, we sent the patients’ oropharyngeal, nasopharyngeal, or sputum samples to the Beijing Center for Disease Prevention and Control. RNA from the viruses was isolated using automated nucleic acid purification (KingFisher Flex Purification System, Thermo, USA). Using 8 μL of input RNA and random hexamers, first-strand cDNA was synthesized using the SuperScript IV First-Strand Synthesis System (Invitrogen, Waltham, MA, USA). Using the ARTIC nCoV-2019 sequencing technique v4.1 (https://github.com/artic-network/artic-ncov2019), 25–32 PCR cycles were performed to generate tiled-PCR amplicons. Primers for pools 1 and 2 were synthesized by Sangon (Shanghai, China). Next-generation sequencing libraries were prepared using the Nextera XT Library Prep Kit (Illumina, San Diego, CA, USA) and sequenced on MiniSeq with 2 × 150 paired-end sequencing kits (Illumina, San Diego, CA, USA). Negative control samples were processed and sequenced in parallel for each sequencing run as contamination controls.

Tian D., Pan Y., Ge Z., Kong X., Zhang Y., Zhang Q., Wang A., Yang P, & Chen Z. (2023). Difference of Clinical Characteristics in Patients with Omicron and Delta Variants of SARS-CoV-2 in Beijing, China. Medicine, 3(2), 75-82.

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