High Pure Viral Nucleic Acid Kit

Rapidly isolate highly purified viral DNA

and RNA
Choose the High Pure Viral Nucleic Acid Kit to efficiently   Use one versatile kit for diverse
isolate viral DNA and RNA from a broad range of research applications.
sample materials, including:
Isolate viral DNA and RNA from a wide variety of sample
Serum, plasma, whole blood, cell culture supernatant,
materials, enabling simultaneous analysis of both virus types.
peripheral blood mononuclear cells (PBMCs), cerebrospinal
fluid (CSF), tongue scrapings, and throat wash samples.
  Save time with a rapid, easy-to-use
­protocol.
Use a simple, rapid protocol to generate high-quality
templates for direct use in PCR, RT-PCR, qPCR, and Prepare multiple PCR/RT-PCR templates in 20 minutes,
qRT-PCR applications. with only 10 minutes of hands-on time.

  Maximize performance and accuracy in

downstream assays.
Experts at Extraction Obtain highly pure, concentrated (50 µl) nucleic acids that
One of the few mammals to use tools, the Sea Otter (Enhydra lutris) uses
provide high sensitivity, reproducibility, and specificity in
rocks or other objects to crack open shellfish to extract its meal. real-time PCR and other applications (Figure 1).

Figure 1: Real-time, on-line PCR detection for the quantification of

Epstein-Barr Virus (EBV) DNA in research samples. EDTA blood samples,
artificially spiked with quantified EBV virus material, were extracted using the
High Pure Viral Nucleic Acid Kit and analyzed using the LightCycler® EBV
Quantification Kit in qPCR on a LightCycler® Instrument. DNA standards
ranging from 106 to 102 copies/reaction (black line with crosses) were used to
generate a standard curve.
EBV-positive blood samples (triplicate, spiked with the EBV genome containing
Namalwa cell line) correspond to the lower limit of 400 copies/reaction (blue
line with dots). The negative control (blood not spiked with Namalwa cell line)
is indicated by the red line with dots.
Results: Efficient nucleic extraction using the High Pure Viral Nucleic Acid Kit
generates purified nucleic acids that do not inhibit the amplification reaction.

www.roche-applied-science.com
Efficiently purify viral nucleic acids High Pure Viral Nucleic Acid Kit workflow

Procedure
Add 200 µl Binding Buffer
200 l serum, plasma, or
Protocol for isolating viral nucleic acids from 200 µl serum, whole blood
supplemented with Poly (A)
and 50 µl Proteinase K
plasma, or whole blood.
Mix immediately and
View detailed procedures for other sample materials in the incubate for 10 min at +72ºC,
then mix s amples with
pack insert at www.roche-applied-science.com 100 µl Binding Buffer

Note: For the isolation of nucleic acids from whole blood,

­pre-warm the elution buffer to +70°C. Combine the High Pure
Filter Tube and the Collection
Tube, then pipet the sample
 To a nuclease-free 1.5 ml microcentrifuge tube: into the upper reservoir
• Add 200 µl serum, plasma, or whole blood.
• Add 200 µl working solution, freshly prepared
(carrier RNA-supplemented Binding Buffer). Centrifuge at 8 ,000 x g
for 1 min
• Add 50 µl Proteinase K solution; mix immediately.
• Incubate for 10 min at +72°C. Add 5 00 µl Inhibitor
Disc ard flowthrough and Removal Buffer
Collection Tube
 Add 100 µl Binding Buffer and mix well. Centrifuge at 8 ,000 x g
for 1 min
 To transfer the sample to a High Pure Filter Tube:
Add 45 0 µl Wash Buffer
• Insert one High Pure Filter Tube into one Collection Tube. Disc ard flowthrough and
Collection Tube
• Pipet the entire sample into the upper reservoir of the Filter Tube. Centrifuge at 8 ,000 x g
for 1 min
 • Insert the entire High Pure Filter Tube assembly into a standard
table-top centrifuge. Add 45 0 µl Wash Buffer
Disc ard flowthrough and
• Centrifuge 1 min at 8,000 × g. Collection Tube
Centrifuge at 8 ,000 x g
for 1 min
 After centrifugation:
• Remove the Filter Tube from the Collection Tube; discard the
flowthrough and the Collection Tube.
Centrifuge at max. speed
• Combine the Filter Tube with a new Collection Tube. (13,000 x g) for 10 seconds

 After combining the Filter Tube with a new Collection Tube: Add 50 µl Elution Buffer
Disc ard flowthrough and
• Add 500 µl Inhibitor Removal Buffer to the upper reservoir of the Filter Collection Tube
Tube. Centrifuge at 8 ,000 x g
for 1 min
• Centrifuge 1 min at 8,000 × g.
 After centrifugation:
• Remove the Filter Tube from the Collection Tube; discard the Purified Viral Nucleic
flowthrough and the Collection Tube. Acids
• Combine the Filter Tube with a new Collection Tube.
 After removal of inhibitors:
• Add 450 µl Wash Buffer to the upper reservoir of the Filter Tube. Ordering information
• Centrifuge 1 min at 8,000 × g and discard the flowthrough.
 After the first wash and centrifugation: Product Cat. No. Pack Size
• Remove the Filter Tube from the Collection Tube; discard the High Pure Viral Nucleic Acid Kit 11 858 874 001 Up to 100 isolations
flowthrough and the Collection Tube.
• Combine the Filter Tube with a new Collection Tube.
• Add 450 µl Wash Buffer to the upper reservoir of the Filter Tube. For more information about the High Pure Viral Nucleic
• Centrifuge 1 min at 8,000 × g and discard the flowthrough. Acid Kit and other products for nucleic acid isolation and
• Leave the Filter Tube-Collection Tube assembly in the centrifuge and
spin it for 10 s at maximum speed (approx. 13,000 × g) to remove any
purification, visit
residual Wash Buffer. www.roche-applied-science.com/napure
T he extra centrifugation time ensures removal of residual Wash
Buffer. HIGH PURE and LIGHTCYCLER are trademarks of Roche.
Other brands or product names are trademarks of their respective holders.
 Discard the Collection Tube and insert the Filter Tube into a nuclease-
free, sterile 1.5 ml microcentrifuge tube.
 To elute the viral nucleic acids:
• Add 50 µl Elution Buffer to the upper reservoir of the Filter Tube. Published by
• Centrifuge the tube assembly for 1 min at 8,000 × g.
 The microcentrifuge tube contains the eluted, purified viral nucleic acids. Roche Diagnostics GmbH
 se the eluted nucleic acids directly in PCR (10 – 20 µl DNA eluate)
U Roche Applied Science
or RT-PCR (3.5 µl viral RNA); or, store the eluted viral RNA at –80°C 68298 Mannheim
or the viral DNA at +2 to +8°C or –15 to –25°C for later analysis. Germany

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