Sand fly DNA was extracted using the Chelex method59 described in Prudhomme et al.27 (link). Extraction was performed at the UMR “Unité des Virus Emergents” (UVE, IHU Méditerranée Infection, Marseille, France). Each entire sand fly was ground using a Mixer Mill MM300 (QIAGEN, Venlo, Netherlands) with one 3-mm tungsten bead in 200 μL Eagle’s Minimal Essential Medium at a frequency of 30 cycles s−1 for 3 min. A volume of 140 μL of each sample was then used for DNA purification by adding the Chelex resin suspension59 or by using the Eppendorf epMotion 5075 working station and the Macherey–Nagel NucleoSpin 96 Virus kit.
Nucleospin 96 virus kit
Manufactured by Macherey-Nagel
Sourced in Germany
The NucleoSpin 96 Virus kit is a laboratory equipment designed for the isolation and purification of viral nucleic acids from various sample types, including cell culture supernatants, swabs, and body fluids. The kit utilizes a silica-membrane based technology to efficiently bind, wash, and elute viral DNA or RNA.
Automatically generated - may contain errors
Lab products found in correlation
Mixer mill mm300, by Qiagen (1 mentions)
7500 real time pcr, by Thermo Fisher Scientific (1 mentions)
Superscript 3 platinum one step quantitative rt pcr system with rox kit, by Thermo Fisher Scientific (1 mentions)
Nuclisens easymag automated system, by bioMérieux (1 mentions)
Biomek nx robot, by Beckman Coulter (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 sybr green 1 master, by Roche (1 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (1 mentions)
Nucleospin 96 tissue kit, by Macherey-Nagel (1 mentions)
Nucleospin 96 rna kit, by Macherey-Nagel (1 mentions)
Nucleospin rna, by Macherey-Nagel (1 mentions)
Sars cov 2 rna, by Twist Bioscience (1 mentions)
Luna universal probe one step rt qpcr kit, by New England Biolabs (1 mentions)
11 protocols using nucleospin 96 virus kit
1
Sand Fly DNA Extraction Protocol
2
Detection of Tick-Borne Encephalitis Virus
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As the TBEV prevalence may be a marker for increased tick activity and the distribution of ticks in space and time, the collected ticks were checked for TBEV-RNA. All collected ticks were examined by two different real-time RT-PCRs for TBEV-RNA: a PCR according to Schwaiger and Cassinotti (2003 (link)) was used in a modified version (Klaus et al. 2010b (link)) and confirmed by an independent TBEV assay (Klaus et al. 2010a (link)). For PCR, the ticks were individually ground in a mixer mill (Retsch, Haan, Germany) with three stainless steel beads and 400 μl medium (MEM Earle, Biochrom AG, Berlin, Germany). Aliquots of the suspensions were pooled (50 μl each from 10 adults/nymphs or 20 larvae) and RNA extracted using the NucleoSpin® 96 Virus kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions on an automated liquid handling station (Star, Hamilton Star, Bonaduz, Switzerland). All RNA samples were stored at − 80 °C to avoid RNA degradation. If a pool was TBEV-positive, RNA was extracted from the individual ticks and checked for the presence of TBEV-RNA. Previous work had demonstrated that it was possible to detect a single TBEV-positive tick in a pool (Klaus et al. 2012 (link)).
3
Quantification of YF-17D RNA in Plasma Samples
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YF-17D RNA was quantified in plasma samples obtained at baseline and on D3, D5, and D7 in the clinical study and on D2, D3, D4, D5, D6, D7, and D10 in the cynomolgus macaque study. Total RNA from plasma samples was extracted using a Nucleospin 96 virus kit (Macherey Nagel cat # 740691) on the TECAN Evoware platform as per the manufacturer’s instruction, and YF-17D RNA copy numbers determined by qRT-PCR based on the detection of the NS5 gene fragment, as described previously (47 (link)). The limit of detection (LOD) and limit of quantification (LOQ) of these assays were 3.0 Log genomic equivalents (GEQ)/mL (i.e., 1 to 10 plaque-forming units (PFU)/mL) and 3.3 Log GEQ/mL, respectively. Samples where no virus was detected were assigned a value of 2.7 Log GEQ/mL (i.e., half the LOD) for geometric mean concentration (GMC) calculations.
4
Dengue Serotyping in Guadeloupe and Martinique
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During the last dengue epidemic of 2019–2021, 3228 available sera from dengue cases confirmed by the IPG, the CHUG, and the CHUM were used. Venous blood samples (5 mL) from symptomatic patients were collected by several laboratories in the frame of their diagnostic activities and stored at 4 °C during the transportation to the laboratories.
In Guadeloupe, serum samples from CHUG and IPG were first submitted to nucleic acid extraction using the NucleoSpin® 96 Virus kit (Macherey Nagel, Duren, Germany) at IPG, following the manufacturer’s instructions, and then screened for presence of DENV by real-time RT-PCR using primers previously described [25 (link)]. If samples were DENV-positive, serotyping was conducted using specific real-time RT-PCR primers [26 (link),27 ]. The real-time RT-PCRs were performed using Superscript® III Platinum One step Quantitative RT-PCR System with ROX kit (Invitrogen, Carlsbad, CA, USA) and the 7500 Real-Time PCR (Applied Biosystems, Waltham, MA, USA).
In Martinique, nucleic acids were extracted at CHUM from plasma using the NucliSens easyMag automated system (BioMérieux, Craponne, France). Samples were then screened by real-time RT-PCR using the SimplexaTM Dengue Kit (DiaSorin Molecular, Cypress, CA, USA) for virus detection and serotype determination.
In Guadeloupe, serum samples from CHUG and IPG were first submitted to nucleic acid extraction using the NucleoSpin® 96 Virus kit (Macherey Nagel, Duren, Germany) at IPG, following the manufacturer’s instructions, and then screened for presence of DENV by real-time RT-PCR using primers previously described [25 (link)]. If samples were DENV-positive, serotyping was conducted using specific real-time RT-PCR primers [26 (link),27 ]. The real-time RT-PCRs were performed using Superscript® III Platinum One step Quantitative RT-PCR System with ROX kit (Invitrogen, Carlsbad, CA, USA) and the 7500 Real-Time PCR (Applied Biosystems, Waltham, MA, USA).
In Martinique, nucleic acids were extracted at CHUM from plasma using the NucliSens easyMag automated system (BioMérieux, Craponne, France). Samples were then screened by real-time RT-PCR using the SimplexaTM Dengue Kit (DiaSorin Molecular, Cypress, CA, USA) for virus detection and serotype determination.
5
RVFV RNA Detection Protocol
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The pooled insects were ground up with 400 µl of PBS 1X (Phosphate Buffered Saline) twice for 30 seconds with two 3-mm diameter stainless beads using the TissueLyser system (Loudet, France) and transferred to a 96-well plate. Total RNA was extracted with the Biomek NX robot (Beckman Coulter, USA) using the NucleoSpin 96 Virus kit (Macherey-Nagel, Germany). For RVFV RNA detection, the L-Segment based SYBR-Green real time PCR was used [27] (link), [28] (link). Ten-fold serial dilutions of a Smithburn strain which contained 108 TCID50/ml were used as the standard curve for plate validation.
6
ZIKV RNA Quantification in Bio-Fluids
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ZIKV RNA in plasma, saliva, ocular fluid and CSF samples from groups A, C, D, and E was quantified post-challenge using a qRT-PCR targeting the NS5 gene22 (link). Total genomic RNA was first extracted from samples with a Macherey Nagel NucleoSpin® 96 virus kit (Macherey Nagel, Germany) on a Tecan Evoware automated RNA-extraction workstation according to the manufacturer’s instructions and eluted in nuclease-free water.
7
Zika Virus Inhibition by Labyrintin Compounds
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Example 7
Cultures of Huh-7.5 cells (human hepatocarcinoma cell line) were treated with various concentrations of either LabyA1, LabyA2 or an equivalent combination of LabyA1 and LabyA2 for 30 min. Cells were subsequently infected with ZIKV (Strain MR766-NIID) at a MOI of 0.5 PFU for 2 h at room temperature. Unbound viral particles were removed and infected cells were incubated for 48 h. Hereafter viral RNA was isolated from the cell culture supernatant (150 μl) using the NucleoSpin® 96 Virus Kit [Macherey-Nagel] according to the vendor's manual. RNA was quantified by absorbance and 2.5 μg were reversely transcribed via RevertAid Reverse Transcriptase [Thermo] with RT-Primer [5′-GGTTTCCCAGCTTCTCCTGG-3′] (SEQ ID NO:5). 100 ng of reverse transcribed RNA were subjected to SYBR-green based quantitative RT-PCR using the LightCycler®480 with LightCycler®480 SYBR Green I Master [Roche] and a ZIKV-specific forward and reverse primer pair (5′-AAAAACCCCATGTGGAGAGG-3′ (SEQ ID NO:6) and 5′-CATTCCTTCAGTGTGTCACC-3 (SEQ ID NO:7)′, respectively). The absolute number of ZIKV-genome copy equivalents (GCE) was determined via standard curves generated from plasmids with known concentrations carrying the respective amplified fragment of the ZIKV genome. Values are ±SEM; n=4. LabyA1 (IC50=4.6 μg/ml); LabyA2 (IC50=5.4 μg/ml); combination LabyA1 and LabyA2 (IC50=3.5 μg/ml).
8
Quantifying HBV Infection in HepaRG and PHH
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Differentiated HepaRG cells (dHepaRG) and PHHs were infected with HBV genotype D (concentrated from HepAD38 cells supernatants) as described previously [20] . After treatment procedures, levels of intracellular and/or extracellular HBV RNAs and DNAs, HBc, secretion of HBeAg and HBsAg were monitored by quantitative polymerase chain reaction (qPCR), reverse transcription qPCR (RT-qPCR), immunoblot and enzyme-linked immunosorbent assay (ELISA) respectively, as previously described [6, 20] . Briefly, HBeAg and HBsAg were quantified in culture medium using a chemiluminescence immunoassay kit (Autobio Diagnostics) according to manufacturer's instructions. Total intracellular DNAs and RNAs as well as secreted viral DNAs/RNAs were purified from infected cells using NucleoSpin® 96 Tissue kit, NucleoSpin® 96 RNA kit, NucleoSpin® 96 virus kit (Macherey-Nagel) respectively, according to manufacturer's instructions. Reverse-transcription of RNA into cDNA was performed with the SuperScript® III First-Strand Synthesis kit (Invitrogen) according to manufacturer's instructions. All qPCR analyses were performed with LightCycler™ 96 and 480 systems (Roche), except for cccDNA and pgRNA specific PCRs that were processed under a QuantStudio™ 7 (Applied), using homemade TaqMan assays [20] .
9
Quantification of CVB3 Viral RNA and Infectious Virions
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Vero A cells were seeded at a density of 5 × 104 cells/well in 96-well tissue culture plates. The next day, cells were treated with serial dilutions of the compound and then infected with CVB3 (MOI, 0.01). After a 2-h incubation, the cells were washed three times with assay medium to remove non-adsorbed virus. The cells were treated with the same serial dilutions of the compound as for the CPE reduction assay and incubated for 2 days at 37°C. At the end of the incubation period, the viral RNA was isolated from the culture supernatants in different wells using NucleoSpin 96 virus kit (Macherey Nagel, Germany) and was quantified by real-time quantitative reverse transcription PCR (qRT-PCR) (see below). In addition, the number of infectious virions in the collected supernatants was determined by an end-point titration assay, as described by Reed and Muench (TCID50/mL) [20 (link)].
10
Viral Infection Dynamics in Cell Cultures
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Vero, C6/36, and Lulo cells were seeded in 12.5-mL flasks (Falcon ® ) using growth media. The number of cells seeded for each cell line was different in order to reach 70-80% confluence during the following 24 h. The growth medium was replaced with 2 mL of assay medium supplemented with 2% instead of 10% FBS containing the appropriate virus dilution. Vero, C6/36, and Lulo monolayers were infected with DENV2 NGC (multiplicity of infection (MOI) of 0.001), YFV (MOI of 0.001), and CHIKV 899 (MOIs of 0.001 and 0.00001). In addition, Lulo cells were infected with DENV2 NGC at a MOI of 0.1 and 0.5. After 2 h of incubation at 20 -22 o C, cell monolayers were washed with phosphate buffered saline (PBS) in order to remove non-adsorbed viruses, and cultures were further incubated. Flasks were monitored daily for cytopathic effect (CPE) development, and cells and supernatants were harvested in all experiments every 48 h, except for CHIKV 899 infected at the lowest MOI, where cells and supernatants were harvested at 6, 16, 28, 40, 52, 64, and 76 h post-infection (p.i.). Total and viral RNA were isolated using the NucleoSpin ® RNA (Macherey-Nagel, Düren) and the NucleoSpin 96 virus kit (Macherey-Nagel), respectively.
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