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Maxwell viral total nucleic acid purification kit

Manufactured by Promega
Sourced in United States

The Maxwell Viral Total Nucleic Acid Purification kit is a laboratory equipment product designed for the purification of viral total nucleic acids from various sample types. The kit utilizes magnetic particle technology to efficiently capture and isolate viral nucleic acids, including both DNA and RNA, from the sample.

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12 protocols using maxwell viral total nucleic acid purification kit

1

Viral Nucleic Acid Extraction and Purification

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Cell cultures were collected at different times following infection and processed using the Maxwell Viral Total Nucleic Acid Purification Kit on a Maxwell Instrument (Promega, Fitchburg, WI, USA), in order to obtain a total nucleic acids fraction in elution volumes of 120 µL. For DNA analysis, an aliquot of the eluted nucleic acids was directly amplified, while for RNA amplification, aliquots of experimental samples were treated with Turbo DNAfree reagent (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) before the amplification step.
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2

Sensitive qRT-PCR Assay for Viral RNA

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Viral loads were measured by Virology Services (WNPRC). vRNA was isolated from plasma samples using the Maxwell Viral Total Nucleic Acid Purification kit on the Maxwell 48RSC instrument (Promega, Madison WI). vRNA was then quantified using a highly sensitive qRT-PCR assay based on the one developed by Cline et al. (48 (link)). RNA was reverse transcribed and amplified using TaqMan Fast Virus 1-Step Master Mix (Invitrogen) on the LightCycler 480 or LC96 instrument (Roche, Indianapolis, IN) and quantified by interpolation onto a standard curve made up of serial ten-fold dilutions of in vitro transcribed RNA. RNA for this standard curve was transcribed from the p239gag_Lifson plasmid, kindly provided by Dr. Jeffrey Lifson, (NCI/Leidos). The final reaction mixtures contained 150 ng random primers (Promega, Madison, WI), 600 nM each primer and 100 nM probe. Primer and probe sequences are as follows: forward primer: 5′- GTCTGCGTCATCTGGTGCATTC-3′, reverse primer: 5′-CACTAGCTGTCTCTGCACTATGTGTTTTG-3′ and probe: 5′-6-carboxyfluorescein-CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG-BHQ1-3′. The reactions cycled with the following conditions: 50°C for 5 min, 95°C for 20 s followed by 50 cycles of 95°C for 15 s and 62°C for 1 min. The limit of detection of this assay is 100 copies/mL.
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3

Quantitative SARS-CoV-2 RNA Measurement

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Viral loads were measured by Virology Services (WNPRC). vRNA was isolated from plasma samples using the Maxwell Viral Total Nucleic Acid Purification kit on the Maxwell 48RSC instrument (Promega, Madison WI). vRNA was then quantified using a highly sensitive qRT-PCR assay based on the one developed by Cline et al. [104 (link)]. RNA was reverse transcribed and amplified using TaqMan Fast Virus 1-Step Master Mix qRT-PCR Master Mix (Invitrogen) on the LightCycler 480 or LC96 instrument (Roche, Indianapolis, IN) and quantified by interpolation onto a standard curve made up of serial ten-fold dilutions of in vitro transcribed RNA. RNA for this standard curve was transcribed from the p239gag_Lifson plasmid, kindly provided by Dr. Jeffrey Lifson, (NCI/Leidos). The final reaction mixtures contained 150 ng random primers (Promega, Madison, WI), 600 nM each primer and 100 nM probe. Primer and probe sequences are as follows: forward primer: 5′- GTCTGCGTCATCTGGTGCATTC-3′, reverse primer: 5′-CACTAGCTGTCTCTGCACTATGTGTTTTG-3′ and probe: 5′-6-carboxyfluorescein-CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG-BHQ1-3′. The reactions cycled with the following conditions: 50°C for 5 min, 95°C for 20 s followed by 50 cycles of 95°C for 15 s and 62°C for 1 min. The limit of detection of this assay is 100 copies/mL.
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4

SIV Infection Quantification in Macaques

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Vaccinated and unvaccinated macaques designated for SIV infection were infected intrarectally with SIVmac239 (3,000 50% tissue culture infectious dose IU). Plasma viraemia was monitored serially by quanititative PCR (qPCR) as previous described64 (link),65 (link). Viral RNA was isolated using the Maxwell Viral Total Nucleic Acid Purification Kit (Promega) and reversed transcribed using the TaqMan Fast Virus 1-Step quantitative reverse transcription PCR (qRT–PCR) Kit (Invitrogen). DNA was quantified on a LightCycler 480 (Roche).
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5

SARS-CoV-2 Detection via RT-qPCR

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RT-qPCR was performed as described previously [10 (link)]. Nucleic acid extraction of SARS-CoV-2 was performed using the Maxwell® Viral Total Nucleic Acid Purification Kit (Promega Corporation, Madison, WI, USA), and RT-qPCR was performed using the One Step PrimeScript™ III RT-qPCR mix (Takara Bio Inc., Japan). The N gene was targeted with a forward primer (2.4 μM), 5′-AAA TTT TGG GGA CCA GGA AC-3′; reverse primer (3.2 μM), 5′-TGG CAG CTG TGT AGG TCA AC-3′; and probe (0.4 μM) 5′-FAM-ATG TCG CGC ATT GGC ATG GA-BHQ-3′. The method was performed according to the manufacturer's protocol.
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6

Plasma Viral Load Quantification

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Plasma was isolated from undiluted whole blood by Ficoll-based density centrifugation and cryopreserved at −80°C. Plasma viral loads were quantified as previously described35 . Briefly, viral RNA (vRNA) was isolated from plasma samples using the Maxwell Viral Total Nucleic Acid Purification kit (Promega, Madison WI). Next, vRNA was reverse transcribed using the TaqMan Fast Virus 1-Step qRT-PCR kit (Invitrogen) and quantified on a LightCycler 480 instrument (Roche, Indianapolis, IN). Primers and probes are described in36 (link).
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7

Quantification of Plasma Viral Loads

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Plasma was isolated from undiluted whole blood by Ficoll-based density centrifugation and cryopreserved at -80°C. Plasma viral loads were quantified as previously described [41 ]. Briefly, viral RNA (vRNA) was isolated from plasma samples using the Maxwell Viral Total Nucleic Acid Purification kit (Promega, Madison WI). Next, vRNA was reverse transcribed using the TaqMan Fast Virus 1-Step qRT-PCR kit (Invitrogen) and quantified on a LightCycler 480 instrument (Roche, Indianapolis, IN). Primers and probes are described in [72 (link)].
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8

Quantifying SIV Viral Load in Plasma

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For animals infected with SIV, plasma viral load was quantified by quantitative reverse transcription-PCR (qPCR) as previously described (90 (link)). In brief, plasma was isolated from whole blood by Ficoll-based density centrifugation. Viral RNA (vRNA) was isolated from cryopreserved plasma samples using the Maxwell Viral Total Nucleic Acid Purification kit (Promega). Then, vRNA was reverse transcribed using the TaqMan Fast Virus 1-Step qRT-PCR kit (Invitrogen) and quantified on a LightCycler 480 instrument (Roche). Primers and probes used for qPCR are described in (91 (link)). qPCR was performed in triplicate for each sample. The limit of detection was established by performing serial dilutions of a known standard.
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9

Sensitive ZIKV RNA Quantification

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Viral RNA was isolated from plasma using the Maxwell Viral Total Nucleic Acid Purification kit on the Maxwell 48 RSC instrument (Promega). Viral RNA was then quantified using a highly sensitive Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay based on the one developed by Lanciotti et al. (2008), though the primers were modified to accommodate both Asian- and African-lineage Zika viruses. RNA was reverse transcribed and amplified using the TaqMan Fast Virus 1-Step Master Mix RT-qPCR kit (LifeTechnologies) on the LightCycler 480 (Roche) and quantified by interpolation onto a standard curve made up of serial 10-fold dilutions of in vitro-transcribed RNA. RNA for this standard curve was transcribed from a plasmid containing an 800-bp region of the Zika virus genome that is targeted by the RT-qPCR assay. The final reaction mixtures contained 150 ng random primers (Promega), 600 nM each primer, and 100 nM probe. Primer and probe sequences are as follows: forward primer: 5′-CGYTGCCCAACACAAGG-3′, reverse primer: 5′-CCACYAAYGTTCTTTTGCABACAT-3′ and probe: 5′−6-carboxyfluorescein-AGCCTACCTTGAYAAGCARTCAGACACYCAA-BHQ1-3′. The reactions cycled with the following conditions: 50°C for 5 minutes, 95°C for 20 seconds followed by 50 cycles of 95°C for 15 seconds, and 60°C for 1 min. The limit of detection of this assay is 150 copies/mL.
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10

Quantifying SHIV Viral Loads in Plasma

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We inoculated the MCMs intravenously with 500 median tissue culture infectious doses (TCID50) of SHIV162P339 (link). Plasma viral loads were measured via quantitative real-time polymerase chain reactions (qRT-PCRs) as previously described70 (link),71 (link). Briefly, vRNA was isolated from plasma using the Maxwell Viral Total Nucleic Acid Purification kit (Promega) on a Maxwell 48 RSC instrument (Promega). Next, vRNA was reverse transcribed into complementary DNA (cDNA), amplified using the TaqMan Fast Virus 1-Step Master Mix qRT-PCR kit (Invitrogen) on a LightCycler 480 or LC96 instrument (Roche), and quantified by interpolation onto a standard curve of tenfold serial dilutions of an SIV gag in vitro transcript. The assay limit of detection was 100 vRNA copies/mL.
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