The wastewater was concentrated using the flocculation method using 3.0% w/v beef extract solution (Sigma-Aldrich, St. Louis, USA), pH 9.5 in 0.05 M glycine buffer. Each sewage water (SW1, SW2, and SW3) sample (500 mL) was acidified (Michael-Kordatou et al. 2020 (link)) at pH 3.5 ± 0.1 and 10 mL of the beef extract was agglomerated by the addition of 0.1 M HCl at pH 3.0 followed by stirring for 10 h. The stirred suspension was centrifuged at 10,000×g for 30 min at 4°C. The pellet was dissolved in 8 mL of phosphate buffered saline (PSB) and the RNA was extracted using NucliSENS® miniMAG® system (BioMerieux, Marcy l Etoile, France) according to the manufacturer’s instructions.
Nuclisens minimag system
Manufactured by bioMérieux
Sourced in France
The NucliSENS® miniMAG® system is a compact and automated nucleic acid extraction and purification platform designed for use in molecular diagnostic laboratories. The core function of this system is to efficiently extract and purify nucleic acids, such as DNA and RNA, from a variety of sample types. The NucliSENS® miniMAG® system utilizes magnetic bead-based technology to capture, wash, and elute the target nucleic acids, providing a reliable and consistent process for sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Beef extract solution, by Merck Group (2 mentions)
Nuclisens lysis buffer, by bioMérieux (2 mentions)
Onestep pcr inhibitor removal kit, by Zymo Research (1 mentions)
Magnetic silica beads, by bioMérieux (1 mentions)
Itaq universal probes one step kit, by Bio-Rad (1 mentions)
Iscript one step rt pcr kit for probes, by Bio-Rad (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Abi 7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Thermomixer, by Eppendorf (1 mentions)
8 protocols using nuclisens minimag system
1
Wastewater Viral RNA Extraction Protocol
2
Wastewater Virus Concentration and RNA Extraction
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The wastewater concentration was conducted by direct flocculation [45 (link)] using beef extract solution (3.0% w/v, pH 9.5; Sigma-Aldrich, St. Louis, USA) in glycine buffer (0.05 M). Briefly, 500 mL of the wastewater sample was acidified (pH 3.5 ± 0.1) and 10 mL of beef extract flocculated by the addition of 1 and 0.1 M HCl (pH 3.5 to 3.0; a visible floc formed) was added. The suspension was stirred for 10 h to allow the viruses present to adsorb to the flocs and subsequently centrifuged at 10,000× g for 30 min at 4 °C. The pellet was dissolved in 8 mL of phosphate-buffered saline (PBS) and RNA isolated with the NucliSENS® miniMAG® system (BioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions.
3
Automated RNA Extraction Using Magnetic Beads
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RNA extraction was performed using a semi-automated method with magnetic silica beads (supplied by bioMerieux, Marcy l’Etoile, France). After an incubation step at room temperature for 20 min, 100 μL of magnetic silica beads were added, and, after a further 10 min incubation, an automated procedure was performed using a nucleic acid purification system (Auto-Pure96, All Sheng Instruments, Zhejiang, China) or a Nuclisens MiniMag system (bioMerieux, Marcy l’Etoile, France). The extracted nucleic acids were then purified from potential PCR inhibitors using the OneStep PCR Inhibitor Removal Kit (Zymo Research, CA, USA).
4
Mosquito Body and Leg Viral RNA Extraction
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Individual mosquito bodies and legs were homogenized with metal beads for 4 min at 20 Hz (Mixer Mill Retsch MM301, Germany) using cell-culture medium supplemented at 20% FBS for bodies and NucliSENS lysis buffer (bioMérieux, France) for legs [19 (link)]. Homogenate supernatants were recovered after centrifugation 5 minutes at 20,000 x g. Nucleic acids were extracted with the NucliSENS miniMAG system (bioMérieux, France) in accordance with manufacturer’s instructions. Real time RT-PCR were performed on a CFX96 Touch Real-Time PCR Detection System instrument using iTaq Universal Probes One-Step Kit (Bio-Rad Laboratories, France) and the primers and probes previously described [4 (link)].
5
Mosquito Viral RNA Extraction and Detection
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Individual mosquito legs and bodies were separately homogenized with metal beads at 20 Hz for 4 min (Mixer Mill Retsch MM301, Germany) either in cell-culture medium supplemented at 20% FBS for bodies or directly in NucliSENS lysis buffer (bioMérieux, France) for legs. Homogenate supernatants were recovered after centrifugation at 20,000 x g during 5 minutes. Viral RNA was extracted using NucliSENS miniMAG system (bioMérieux, France) according to manufacturer’s instructions. Real time RT-PCR was processed on a CFX96 Touch Real-Time PCR Detection System instrument using iScript One-Step RT-PCR Kit for Probes (Bio-Rad Laboratories, France). The primers and the probe used were previously described [30 (link)].
6
Quantifying ZIKV RNA in Biological Fluids
7
DNA Extraction from Blood and Tissue
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Chewed plant samples were processed according to previously described techniques38 (link). DNA was extracted from whole blood samples using DNEasy® blood and tissue kits (Qiagen, Valencia, CA, USA). Total nucleic acid was extracted from approximately 100 ug of frozen tissue. Tissue samples collected at necropsy were cut into small pieces with scissors disinfected with 10% sodium hypochlorite after handling each sample, to prevent DNA cross-contamination between tissues and between animals. Total nucleic acid was extracted using the NucliSENS® MiniMAG® system (bioMérieux, Inc.). DNA and total nucleic acid were stored at −80 °Celsius.
8
Viral RNA Extraction from Magnetic Beads
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The suspension of ApoH magnetic beads in lysis buffer was vortexed, held at 56 °C for 30 min, and then centrifuged at 1800× g for 2 min. Viral RNA was then extracted according to the procedure recommended by the manufacturer of the NucliSENS® miniMAG® system (bioMérieux), eluted in 100 µL of NucliSENS® elution buffer 3, stirred at 1400 rpm for 5 min at 60 °C in a thermomixer (Eppendorf, Hamburg, Germany), and then held for 1 min on a magnetic stand (bioMérieux). The supernatant was stored at −80 °C until RT-qPCR analysis.
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