Nasopharyngeal swabs or bronchoalveolar lavage fluids (BALFs) were tested for SARS-CoV- 2 by real-time qPCR. The tests were performed in the Laboratory of High-Performance Molecular Diagnostic or Laboratory of Clinical Pathology, all located at the University of Campinas. The samples were aliquoted and extracted manually using the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Applied Biosystems). Some RNAs were automatically isolated by MagMAX Express-96 Magnetic Particle Processor (Applied Biosystems) following the manufacturer’s protocol. Real-time qPCRs were performed in duplicates using TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems) for the detection of SarbecoV E-gene with specific primers (10 µM) and probe (5 µM). Primer sequences were: forward-ACAGGTACGTTAATAGTTAATAGCGT ; reverse-ATATTGCAGCAGTACGCACACA , Probe:6FAM-ACACTAGCCATCCTTACTGCGCTTCG - QSY. Patients were considered positive for COVID-19 when Ct < 38. qPCRs were performed using the Applied Biosystems 7500 Fast Real-time PCR system.
Magmax express 96 magnetic particle processor
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
The MagMAX™ Express-96 Magnetic Particle Processor is a fully automated instrument designed for high-throughput nucleic acid purification. It utilizes magnetic particle technology to extract and purify DNA, RNA, or proteins from a variety of sample types. The processor can handle up to 96 samples simultaneously, providing efficient and consistent results.
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Lab products found in correlation
Magmax pathogen rna dna kit, by Thermo Fisher Scientific (4 mentions)
Magmax 96 viral rna isolation kit, by Thermo Fisher Scientific (4 mentions)
Taqman fast virus 1 step master mix, by Thermo Fisher Scientific (3 mentions)
Magmax total rna isolation kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Magmax viral pathogen nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Quantifast probe pcr kit, by Qiagen (1 mentions)
Applied biosystems 7500 real time pcr instrument, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr instrument, by Thermo Fisher Scientific (1 mentions)
Agpath id one step rt pcr reagent, by Thermo Fisher Scientific (1 mentions)
Cfx96 touch, by Bio-Rad (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Magmax 96 total rna isolation kit, by Thermo Fisher Scientific (1 mentions)
15 protocols using magmax express 96 magnetic particle processor
1
SARS-CoV-2 Detection in Clinical Samples
2
Nucleic Acid Extraction from Lung Tissues
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For nucleic acid extraction, the MagMAX™ Pathogen RNA/DNA Kit (Life Technologies, Carlsbad, CA, USA) and the MagMAX™ Express-96 Magnetic Particle Processor (Applied Biosystems, Foster City, CA, USA) were used following the manufacturer's instructions. For the lung tissues bead-beating method was used as a preparative step prior to DNA extraction. A NanoDrop® ND-1000 spectrophotometer was used to quantify and verify the quality of the extracts.
3
Abalone Tissue Nucleic Acid Extraction
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Nucleic acid from AbHV-challenged and -unchallenged control abalone tissues (approximately 100 mg of tissue including the target neural tissue surrounded by some muscle) was extracted (using Applied Biosystems Mag MAX Express 96 Viral Isolation Kit and the MagMAX Express-96 Magnetic Particle Processor) following the manufacturer's instructions, except that the final elution was made in 50 µl.
4
Tissue RNA Extraction for Pathogen Analysis
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Liver, spleen, kidney, GIT, brain and blood specimens were collected on days 1, 4, 6 and 7 post-infection, and from animals reaching experimental end points. RNA was extracted using MagMax Total RNA Isolation kit (Ambion). Approximately 100 mg tissue samples were placed directly in lysis buffer and homogenized using a high throughput tissue grinder (GenoGrinder2000). Homogenates were extracted using the MagMax Express-96 Magnetic Particle Processor (Ambion) according to manufacturer's directions including a DNase treatment step. Approximately 50 mL of whole blood was added directly to lysis buffer with isopropanol and extracted using the MagMax Express-24 Magnetic Particle Processor (Ambion) following manufacturer's protocol.
5
Comprehensive Tissue RNA Extraction
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Mice were deeply anesthetized with isofluorane, terminally bled and perfused with 10 mL PBS. Specimens of olfactory bulb, cerebral cortex, cerebellum, brainstem, liver, spleen, popliteal lymph node, sciatic nerve and whole blood were collected. RNA was extracted using MagMax Total RNA Isolation kit (Ambion). With the exceptions of the sciatic nerve and popliteal lymph node, in which cases the entire structures were used, approximately 100 mg of tissue were placed directly in lysis buffer and homogenized using a high-throughput tissue grinder (GenoGrinder2000). Approximately 50 µL of whole blood was added directly to lysis buffer with isopropanol. Homogenates were extracted using the MagMax Express-96 Magnetic Particle Processor (Ambion) according to manufacturer's directions including a DNase treatment step.
6
SUDV Detection in Serum Samples
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Total RNA was purified from serum samples by using the MagMAX-96 Viral RNA Isolation Kit on the MagMAX Express-96 Magnetic Particle Processor (Ambion, Grand Island, NY, USA). In parallel, RNA was purified from serial dilutions of serum samples from healthy persons that were inoculated with a known titer of SUDV and used to generate a standard curve. Real-time PCR was then performed as previously described (12 (link)).
7
Fecal Nucleic Acid Extraction and Viral Detection
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Nucleic acid was extracted from faecal samples using an RNA-DNA isolation kit (MagMAX Pathogen RNA/DNA Kit; Life Technologies GmbH, Darmstadt, Germany). Briefly, 0.5 g of faeces was suspended in 1 mL PBS and vortexed vigorously for 3 min, until the solution was fully suspended. For a separation of phases, it was centrifuged at 100 x g for 3 to 5 s. A total of 200 μL of the solution was transferred to a tube with 500 μL prepared lysis/binding solution of the RNA-DNA isolation kit. It was vortexed vigorously for 5 min and then centrifuged at 16.000 x g for 3 min to clarify the lysate. Nucleic acid isolation was performed with an automated nucleic acid isolation processor (MagMAX™ Express-96 Magnetic Particle Processor; Life Technologies GmbH) based on magnetic bead technology, according the manufacturer’s protocol and instructions. Extracted nucleic acid was analysed by PCR methodologies.
RVA RNA was detected by a previously described real-time RT-PCR [29 (link)]. PEDV and TGEV were detected by a real-time multiplex RT-PCR for the simultaneously detection of both viruses, following manufacturer recommendations (virotype PEDV/TGEV RT-PCR Kit, Qiagen, Denmark).
RVA RNA was detected by a previously described real-time RT-PCR [29 (link)]. PEDV and TGEV were detected by a real-time multiplex RT-PCR for the simultaneously detection of both viruses, following manufacturer recommendations (virotype PEDV/TGEV RT-PCR Kit, Qiagen, Denmark).
8
Molecular Detection of Mycoplasma Pathogens
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Tonsillar swabs were transported to the laboratory and stored at -20°C until use. DNA was extracted using MagMAX-96 Viral RNA isolation kit and MagMAX Express-96 Magnetic Particle Processor (Life Technologies, Grand Island, NY, USA). For M. hyorhinis genetic material detection, a real-time PCR was performed with QuantiFast Probe PCR kit (Qiagen Inc., Germantown, MD, USA), according to manufacturer’s protocol, employing custom primers and probe [23 (link)]. Similarly, a real-time PCR assay was performed to detect M. hyosynoviae with Path-ID qPCR Master Mix Kit (Life Technologies, Grand Island, NY, USA), according to manufacturer’s protocol. The primers and probes were synthetized based on the Standard Operating Procedures (SOP) routinely followed at the University of Minnesota, Veterinary Diagnostic Laboratory for M. hyosynoviae detection (UMN-VDL SOP .0078). Samples were considered positive by real-time PCR for M. hyorhinis and M. hyosynoviae when Ct ≤ 37.
9
Quantification of African Swine Fever Virus
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Total nucleic acid was extracted from the whole blood and meat exudate using the MagMAX™ Pathogen RNA/DNA Kit (Life Technologies, Burlington, ON, Canada) and the MagMAX Express-96 Magnetic Particle Processor (Life Technologies) following the manufacturer’s protocol. For the organ samples, nucleic acid extraction was performed on supernatant collected from 10% w/v suspensions in sterile PBS. The ASFV genomic material in the whole blood, organs, and meat exudate was quantified using a previously published TaqMan qPCR assay that specifically amplifies a conserved region of the p72 gene of the virus [45 (link)]. The ASFV qPCR was carried out using the TaqMan™ Fast Virus 1-Step master mix (Life Technologies) on the Applied Biosystems 7500 Real-Time PCR Instrument (Life Technologies) using the cycling conditions recommended for the master mix (50 °C for 10 min, 95 °C for 3 min followed by 40 cycles of 96 °C for 3 sec and 60 °C for 30 sec). The TaqMan RT-qPCR assay for beta-actin developed in-house was used to ensure valid nucleic acid extraction and the absence of PCR inhibitors in the samples [46 (link)]. Samples with Ct values of 35.99 and lower were considered positive, and values between 36 and 40 were considered suspicious.
10
FMDV RNA Quantification by RT-qPCR
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The FMDV RNA levels in serum, nasal and oral swabs were quantified by a TaqMan RT-qPCR assay as described previously (22 (link)). Viral RNA was extracted from 50 μl of sample with the MagMAX™-96 Viral RNA Isolation Kit (Life Technologies) using the MagMAX™ Express-96 Magnetic Particle Processor (Life Technologies). One-step RT-qPCR was performed using the AgPath ID One-Step RT-PCR reagents (Life Technologies) on the Applied Biosystems 7500 Real-Time PCR Instrument. All samples were tested in duplicate and samples with poor Ct value correlation in the duplicate reactions were repeated. Samples with a Ct <40 (equivalent to 1 × 103.5 copies RNA/ml blood or 1 × 103.2 copies RNA/swab) were considered positive (23 (link)).
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