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Kinovett Scientific

NEB Monarch® Mag Viral DNA/RNA Extraction Kit (T4010)

NEB Monarch® Mag Viral DNA/RNA Extraction Kit (T4010)

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The Monarch Mag Viral DNA/RNA Extraction Kit enables reliable, high-throughput purification of viral nucleic acid using a magnetic bead-based protocol.

  • Designed for hands-free extraction of viral DNA and/or RNA.
  • Utilizes magnetic bead-based methods. Compatible with manual and automated high-throughput workflows on a variety of instrument platforms, such as KingFisher® Flex, Agilent® Bravo®, MGISP® liquid handlers, and more.
  • Tested for saliva and respiratory swab sample types. Compatible with wastewater samples, after enrichment steps (not supplied).
  • Suitable for qPCR/RT-qPCR, ddPCR, library prep for sequencing/NGS and other downstream applications.
  • Includes carrier RNA for sensitive detection in RNA-based amplification workflows.

Package Sizes:

Catalog # Concentration Size
T4010S Not Applicable 100 preps
T4010L Not Applicable 600 preps
T4010X Not Applicable 1,800 preps


 

The Monarch Mag Viral DNA/RNA Extraction Kit provides a rapid and reliable magnetic bead-based process for extracting viral nucleic acids from saliva and respiratory swab samples. The kit combines the efficiency of silica-based nucleic acid purification with the ease of use of magnetic beads. Manual and automated workflows allow samples to be processed in microfuge tubes or 96-well plates. Kit sizes align to 96-well formats (100 preps, 600 preps, and 1800 preps), and the protocol is compatible with high throughput automation on a variety of platforms, including the KingFisher Flex magnetic particle processor and Agilent Bravo and MGISP liquid handler platforms.

Properties
Purification format Magnetic bead
Processing format Manual or automated
Sample purification (representative examples) Viral DNA and RNA* from respiratory viruses (enveloped and non-enveloped, dsDNA and ssRNA)
Sample sources Saliva, respiratory swab in viral transport media (VTM)**
Sample input volume Up to 200 μl**
Carrier supplier Poly A carrier RNA***
Binding capacity Up to 3 μg
Elution volume 33–100 μl
Tested automation platforms KingFisher Flex; Agilent Bravo and MGISP liquid handlers
Compatible downstream applications qPCR, RT-qPCR, ddPCR, library prep for NGS

* Viral DNA and RNA are purified in parallel. Preparation of DNA-free RNA or RNA-free DNA requires further treatment with the appropriate nuclease (not supplied).
** The sample input volume may be scalable to accommodate larger sample volumes. Further workflow optimization may be required.
*** Use of carrier RNA is recommended for recovery of low amounts of viral nucleic acid. Carrier RNA should be omitted if the downstream application utilizes poly(A) RNA enrichment; however, viral nucleic acid recovery may be reduced.

 

Figure 1: Monarch Mag Viral DNA/RNA Extraction Kit Workflow

T4010 Workflow

Overview of workflow of the Monarch Mag Viral DNA/RNA Extraction Kit for manual or automated magnetic bead-based purification of viral nucleic acid.

 
Silica-coated Monarch Mag Beads M1 are utilized for highly sensitive nucleic acid capture from samples containing few target molecules. Silanol groups on the bead shell provide an optimal binding surface for nucleic acids, and the uniform, submicron bead size offers a high surface area with abundant binding sites. Beads are supplied as a monodispersed suspension. The superparamagnetic properties of the bead core result in a fast magnetic response, contributing to ease of handling during use and compatibility with automation

An optimized viral nucleic acid extraction procedure employs a sample lysis step followed by a simple bind-wash-elute process. Samples are treated with Proteinase K and then mixed with a lysis buffer/bead mixture containing lysis buffer, carrier RNA, isopropanol, and magnetic beads, which promote binding of viral nucleic acid onto the silica-coated beads. Carrier RNA is utilized as a co-precipitant in the workflow to enhance the recovery of low amounts of viral nucleic acid. After binding of viral DNA and RNA to the magnetic beads, the beads are washed to remove contaminants, and nucleic acid is eluted in nuclease-free water. Purified viral nucleic acid is suitable for downstream applications, including qPCR/RT-qPCR, ddPCR, and library prep for Next Generation Sequencing (NGS).

Figure 2: Complete SARS-CoV-2 genome coverage from wastewater samples using the Monarch Mag Viral DNA/RNA Extraction Kit

T4010 Genome Coverage

Integrative Genome Viewer visualization of read coverage across the SARS-CoV-2 genome (log scale). RNA was extracted from 750,000 copies of inactivated SARS-CoV-2 viral particles (ATCC® 1986HK) spiked into either the RNA extraction lysis buffer or wastewater samples, using the Monarch Mag Viral DNA/RNA Extraction Kit. Viral particle enrichment was applied to the contrived wastewater samples with Nanotrap® Microbiome A Particles prior to RNA extraction. 20,000 copies of Twist Bioscience Synthetic SARS-CoV-2 RNA Control 23 template served as a positive control for amplicon generation and library prep. Amplicons and libraries were prepared with NEBNext® VarSkip Short v2 SARS-CoV-2 primer pools and the NEBNext ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina). Libraries were sequenced on a NextSeq®500/550 instrument (2x75 bp). Coverage depth per base was determined, reads were down-sampled with seqtk and aligned to SARS-CoV-2 reference genome (NCBI, NC_045512) with Bowtie2.


Figure 3: Performance comparison of Monarch Mag Viral DNA/RNA Extraction Kit with other suppliers demonstrates high reproducibility and sensitivity of the Monarch kit

T4010 CT Comparison

Mock samples representing decreasing viral loads were prepared using Heat-inactivated SARS-CoV-2 (ATCC) in VTM (Hardy Diagnostics®). Extraction was performed using Monarch Mag Viral DNA/RNA Extraction Kit and similar kits from two other suppliers. RT-qPCR was performed using NEB #E3019 and BioRad CFX96 Touch Real-Time PCR Detection System. Monarch Mag Viral DNA/RNA Extraction Kit showed consistently low Cts and reproducible data, even at low viral loads, compared to the competitor kits tested.


Figure 4: qRT-PCR on DNA extracted using the Monarch Mag Viral DNA/RNA Extraction Kit demonstrates accurate detection and quantification of target DNA viruses
T4010 Accurate Detection

Monarch Mag Viral DNA/RNA Extraction Kit was used to extract DNA from i. Human Adenovirus- Type 3 (ZeptoMetrix) and ii. Cytomegalovirus (ZeptoMetrix®) at two different viral loads using the automated KingFisher protocol. The eluted DNA was subjected to qRT-PCR on a Bio-Rad® CFX96 Touch™ Real-Time PCR Detection System. Amplification curves demonstrate successful extraction of viral DNA with expected signal corresponding to the respective viral loads.


Figure 5: RT-qPCR on RNA extracted using the Monarch® Mag Viral DNA/RNA Extraction Kit demonstrates detection of multiple viruses from the same sample

T4010 and FluSARS

Mock co-infection samples representing high and low viral loads were prepared using a swab containing inactivated Influenza and SARS-CoV-2 viruses (Microbiologics®) resuspended in VTM (Hardy Diagnostics®). Extraction was performed using Monarch® Mag Viral DNA/RNA Extraction Kit following the automated KingFisher® protocol. RT-qPCR was performed using relevant primer probes and Bio-Rad® CFX96 Touch™ Real-Time PCR Detection System. Monarch Mag Viral DNA/RNA Extraction Kit can successfully extract nucleic acids from multiple viruses present in a sample.

 

 

 

Product Name: NEB Monarch® Mag Viral DNA/RNA Extraction Kit (T4010)

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