For highly sensitive detection of viral RNA and/or DNA
High sensitivity in single and multiplex assays
Detection of viral RNA and/or DNA in the same reaction
Clear detection of weak positive signals
Fast universal 2-step protocols
5x master mix for higher sensitivity with more sample input
The QuantiTect Virus Kit is specially designed for highly sensitive detection of up to 4 viral nucleic acid targets by multiplex, real-time one-step RT-PCR using sequence-specific probes. Multiple viral RNA and/or DNA targets plus internal controls can be detected in a single tube without loss of sensitivity. The concentrated 5x master mix enables the addition of higher sample input and contains HotStarTaq Plus DNA Polymerase. The QuantiTect Virus RT Mix contains a unique formulation of Sensiscript Reverse Transcriptase that is optimized for highly sensitive detection of viral RNA. Two kit formats are available: the QuantiTect Virus Kit for cyclers that require ROX dye for fluorescence normalization and the QuantiTect Virus +ROX Vial Kit for other cyclers. For convenience, the master mix can be stored at 2–8°C.
For 1000 x 50 µl reactions: QuantiTect Virus NR Master Mix (without ROX dye), ROX Dye Solution, QuantiTect Virus RT Mix, RNase-Free Water, QuantiTect Nucleic Acid Dilution Buffer
QuantiTect Virus Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
Unambiguous determination of CT values over a wide dynamic range.
Serial dilutions of bovine viral diarrhea virus (BVDV) RNA, as indicated, were amplified and analyzed using the QuantiTect Virus Kit. The steep sigmoidal curves enable accurate CT value determination, even at low template amounts (10 copies/μl). [A] Detection of BVDV1 RNA. [B] Detection of BVDV2 RNA. NTC: No template control.
Reliable detection of viral RNA over a wide linear range.
Viral RNA was diluted in serial fivefold dilutions and amplified in duplex with an internal control using the QuantiTect Virus Kit. [A] Amplification plot of the viral targets. [B] Amplification plot of the internal control.
Improved detection of low amounts of viral RNA.
Viral RNA was diluted in serial fivefold dilutions and amplified in duplex with an internal control using the QuantiTect Virus Kit (see figure "Reliable detection of viral RNA over a wide linear range") or kits from Suppliers AII and I. The QuantiTect Virus Kit provided much higher sensitivity than the other reagents tested, enabling reliable analysis of unknown samples. ND: Not detected after 50 PCR cycles.
Performance
Amplification using QuantiTect Virus Kits provides steep sigmoidal curves over a range of dilutions, even for low template amounts with high CT values (see figure "Unambiguous determination of CT values over a wide dynamic range"). This enables accurate CT value determination for quantification of viral nucleic acids in real-time PCR.
QuantiTect Nucleic Acid Dilution Buffer, supplied with the kits, stabilizes RNA and DNA standards during dilution and reaction setup and prevents loss of nucleic acids on plastic surfaces, such as tubes or pipet tips. The buffer enables reliable dilution of standards used to quantify viral nucleic acids, giving a wide linear range, from low to high CT values and ensures longer storage of standards without degradation (see figure "Reliable dilution and storage of RNA standards").
Principle
QuantiTect Virus Kits provide highly sensitive detection of viral nucleic acids in single or multiplex assays on the first attempt (see flowchart "QIAGEN multiplex kits"). The optimized master mix ensures that PCR products in a multiplex reaction are amplified with the same efficiency and sensitivity as PCR products in a corresponding single-amplification reaction.
Amplifying control and target genes in the same reaction, instead of separate reactions, increases the reliability of gene quantification by minimizing handling errors. The QuantiTect Virus Buffer contains a balanced combination of K+ and NH4+ ions as well as unique synthetic Factor MP stabilizes, which together promote stable and efficient annealing of primers and probes to the nucleic acid template, enabling high PCR efficiency (see figure "Unique PCR buffer"). In addition, the unique formulation of Sensiscript Reverse Transcriptase ensures highly sensitive reverse transcription of viral RNA, while HotStarTaq Plus DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.
Components of the QuantiTect Virus Kit
Kit component
Feature
Benefits
5x QuantiTect Virus Master Mix
Concentrated master mix
Highly concentrated and optimized for sensitive virus detection
Larger volumes of template can be added to the assay for increased sensitivity
HotStarTaq Plus DNA Polymerase
5 min activation at 95ºC
Set up of qPCR reactions at room temperature
QuantiTect Virus Buffer
Balanced combination of NH4+ and K+ ions
Specific primer annealing ensures reliable PCR results
Synthetic Factor MP
Reliable multiplexing analysis of up to 4 genes in the same tube
Additional kit components
QuantiTect Virus RT Mix
Contains a unique formulation of Sensiscript Reverse Transcriptase
Optimized for highly sensitive detection of viral RNA
QuantiTect Nucleic Acid Dilution Buffer
Proprietary buffer formulation for dilution and storage of nucleic acid standards.
Stabilizes RNA and DNA standards during dilution and reaction setup and prevents loss of nucleic acids on plastic surfaces, such as tubes or pipet tips
Procedure
QuantiTect Virus Kits provide highly sensitive real-time PCR analysis of viral nucleic acids (RNA and/or DNA) and internal controls using sequence-specific probes. Reactions can be carried out with or without a reverse-transcription step, enabling flexible design of multiplex assays to detect RNA targets, DNA targets, or both RNA and DNA targets. Follow the protocol in the handbook for fast and reliable results.
Kits are available with or without ROX passive reference dye in the master mix (see table).
Choosing the right QuantiTect Virus Kit
ROX dye
Kit
Compatible cyclers
Supplied in master mix
QuantiTect Virus Kit
All cyclers from Applied Biosystems except Applied Biosystems 7500
Supplied in separate tube
QuantiTect Virus +ROX Vial Kit
Applied Biosystems 7500 and cyclers from Bio-Rad, Cepheid, Eppendorf, QIAGEN, Roche, Agilent, and other suppliers
For fast and highly sensitive end-point one-step RT-PCR applications, including virus detection, we recommend using the QIAGEN OneStep RT-PCR Kit.
Applications
QuantiTect Virus Kits provide highly sensitive real-time single or multiplex PCR or one-step RT-PCR using sequence-specific probes for detection of viral DNA and/or RNA plus internal controls. The kits can be used on a wide range of real-time thermal cyclers, including instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, QIAGEN, Roche (except for capillary cyclers), and Agilent.
Features
Specifications
Applications
Virus detection
Reaction type
Reverse Transcription and PCR
Real-time or endpoint
Real-Time
Sample/target type
RNA and/or DNA targets
Single or multiplex
Single or multiplex
SYBR Green I or sequence-specific probes
Sequence-specific probes
Thermal cycler
Most real-time cyclers (except capillary cyclers e.g. LightCycler® 1.x and 2.0)
With or without ROX
Available with ROX in master mix and ROX as a separate vial
For 100 x 50 µl reactions: QIAGEN OneStep RT-PCR Enzyme Mix (1 x 200 µl), 5x QIAGEN OneStep RT-PCR Buffer (1 x 1 ml), dNTP Mix (1 x 200 µl, 10 mM each), 5x Q-Solution (1 x 2 ml), RNase-Free Water (2 x 1.9 ml)
For 100 x 50 µl multiplex PCR reactions: 2x QIAGEN Multiplex PCR Master Mix (providing a final concentration of 3 mM MgCl2, 3 x 0.85 ml), 5x Q-Solution (1 x 2.0 ml), RNase-Free Water (2 x 1.7 ml)
For 25 x 50 µl reactions: QIAGEN OneStep RT-PCR Enzyme Mix (1 x 50 µl), 5x QIAGEN OneStep RT-PCR Buffer (1 x 250 µl), dNTP Mix (1 x 50 µl, 10 mM each), 5x Q-Solution (1 x 400 µl), RNase-Free Water (1 x 1.9 ml)
