Magmax viral pathogen 2 mvp 2 nucleic acid isolation kit

Hellenic Pasteur Institute: Viral RNA was extracted from 200 μl of clinical samples using the NucliSens easyMAG automated system (BioMérieux, Marcy l’Etoile, France). A 106 bp fragment of the SARS-CoV-2 virus RdRp gene was amplified according to an in-house qPCR protocol proposed by the WHO and the National Reference Center for Respiratory Viruses, Institut Pasteur, Paris. As a confirmatory assay, the E gene assay from the Charité protocol was used⁷⁵ by real-time RT-PCR. SeraCare’s SARS-CoV-2 AccuPlex solution 5000copies/ml (Material number 0505–0126) was used for limit-of detection testing. 1 μl of the solution was directly placed in the LAMP mix or after 10 min of heating at 80 °C.
Laboratory of Clinical Virology, Un. of Crete: Viral RNA was extracted from 200 μl of VTM using the MagMAX™ Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit (Applied Biosystems) on the KingFisher™ Flex Purification System (Thermo Scientific) according to the manufacturer’s protocol. QPCR COVID-19 detection was perfomed using the TaqPath COVID-19 CE-IVD RT-PCR kit (Applied Biosystems) on the QuantStudio 5 real-time PCR (Applied Biosystems) with the associated Applied Biosystems COVID-19 Interpretive Software. The TaqPath COVID-19 RT-PCR kit targets genomic fragments in the ORF gene, S gene and the N gene. It uses internal control for assessing extraction efficiency and PCR inhibitors.

The MagMax Viral/Pathogen II (MVP II) Nucleic Acid isolation kit (Applied Biosystems, Foster City, CA) was used to extract nucleic acid (NA) from 200 μL of each specimen. A negative extraction control containing 200 μL viral transport media (Corning, Corning, NY) was included on every plate. Extractions were processed on the Kingfisher Flex Magnetic Particle Processor (Thermo Fisher Scientific Inc., Waltham, MA). The resulting elution volume was 50 μL and the same elution was used for testing with the EZ-SARS-CoV-2 RT-PCR and the TaqPath COVID-19 Combo Kit Multiplex Real-Time RT-PCR assays.

The MagMax Viral/Pathogen II (MVP II) Nucleic Acid isolation kit (Applied Biosystems, Foster City, CA) was used to extract nucleic acid (NA) from 200 μL of each specimen. A negative extraction control containing 200 μL viral transport media (Corning, Corning, NY) was included on every plate. Extractions were processed on the Kingfisher Flex Magnetic Particle Processor (Thermo Fisher Scientific Inc., Waltham, MA). The resulting elution volume was 50 μL and the same elution was used for testing with the EZ-SARS-CoV-2 RT-PCR and the TaqPath COVID-19 Combo Kit Multiplex Real-Time RT-PCR assays.

The MagMAX viral/pathogen II (MVP II) nucleic acid isolation kit (Applied Biosystems) is a nucleic acid purification kit based on magnetic bead technology on the market. Extraction of viral nucleic acid was conducted on the KingFisher Flex Magnetic Particle Processor with a 96 Deep-Well Head (Thermo Scientific) according to kit instructions. Sample volumes were 200 µL, with elution volumes of 100 µL.

For routine analysis, nasopharyngeal specimens in universal transport medium (UTM) were opened under biosafety hood, and 200 μL were used for RNA extraction using MagMAX Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit and the automated KingFisher magnetic particle processor (Thermo Fisher Scientific, Waltham, MA, USA), as indicated in the manufacturer’s instructions. Then, 10 μL of the extracted RNA underwent real-time reverse transcription polymerase chain reaction (qRT-PCR) using the TaqPath™ COVID-19 CE-IVD RT-PCR Kit assay (Thermo Fisher Scientific, Waltham, MA, USA) following the kit’s instruction. The QuantStudio 5 Real-Time PCR System assay (DX) was used for qRT-PCR analysis (Thermo Fisher Scientific, Waltham, MA, USA). The TaqPath™ assay targets three different viral genomic regions: ORF1ab, N and S genes. A valid negative result for SARS-CoV-2 was determined by amplification of the only MS2 internal control. A specimen was considered positive in the presence of amplification of at least two of the three target genes. Particularly, positive samples were classified as “S positive” when all the three targets were detected or when only two targets were detected, including S. On the other hand, samples were defined as “S negative” when the S target was not amplified.

Nasal swabs were collected and placed in 3mL phosphate buffered saline. RNA was extracted from 190 uL of sample using the MagMAX™ Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, MA) and eluted in a volume of 50 μL according to manufacturer’s instructions. 5μL of RNA was quantitated using a one-step RT-PCR using a TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific).

Pools consisting of five samples were constructed from one positive sample and four negative samples. Positive samples were selected at random from -80°C storage and thawed at 2–8°C. RNA extraction was performed using 400 μL of combined sample volume using the MagMAX™ Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit (cat: A48383; Thermo Fisher Scientific, Waltham, MA) by combining 10 μL of Proteinase K, 80 μL of each sample (400 μl pooled volume), 550 μL of lysis buffer with RNA binding beads, and 10 μL of MS2 phage extraction control to a single well of an Agilent 1 mL 96-well plate. The plate was vortexed 2 minutes at 3 x g, incubated 5 minutes at 65°C, vortexed for 5 minutes at 3 x g, and incubated at room temperature on a magnetic stand. After waste aspiration, the beads underwent three cycles of resuspension/aspiration in 1 mL wash buffer, 1 mL 80% ethanol, and 50 μL elution buffer, respectively. RT-qPCR was performed using 17.5 μL of eluant added to 7.5 μL of reaction mix that contained 6.25 μL TaqPath™ 1-Step RT-qPCR MM, CG (cat: A15299; Thermo Fisher, Waltham, MA) and 1.25 μL TaqMan™ SARS-CoV-2 Pooling Multiplex Assay (cat: A50790; Thermo Fisher, Waltham, MA).