QuantiTect Virus Kits

For highly sensitive detection of viral RNA and/or DNA

S_1084_5_GEN_V2

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QuantiTect Virus Kit (1000)

Cat. No. / ID:  211015

For 1000 x 50 µl reactions: QuantiTect Virus Master Mix (contains ROX dye), QuantiTect Virus RT Mix, RNase-Free Water, QuantiTect Nucleic Acid Dilution Buffer
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DKK 40,095.00
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Reference dye
Rox in Mastermix
Rox in vial
Reactions
1000
200
50
QuantiTect Virus Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • 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

Product Details

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.

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.

Multiplex assays enable the detection of multiple viral RNA and/or DNA targets plus internal controls over a wide linear range without loss of sensitivity (see figures " Reliable detection of viral RNA over a wide linear range" and "   Improved detection of low amounts of viral RNA").

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").

See figures

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 componentFeature 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
See figures

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 dyeKit 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.

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsVirus detection
SYBR Green I or sequence-specific probesSequence-specific probes
Real-time or endpointReal-Time
Reaction typeReverse Transcription and PCR
Thermal cyclerMost real-time cyclers (except capillary cyclers e.g. LightCycler® 1.x and 2.0)
Sample/target typeRNA and/or DNA targets
With or without ROXAvailable with ROX in master mix and ROX as a separate vial
Single or multiplexSingle or multiplex

FAQ

How do I setup and validate a multiplex PCR assay with QIAGEN PCR kits?

Ensure PCR amplicons are as short as possible, ideally 60–150 bp. Always use the same algorithm or software to design the primers and probes. For optimal results, only combine assays that have been designed using the same parameters.

 

Check the functionality of each set of primers and probes in individual assays before combining the different sets in the multiplex assay. Choose compatible reporters and quenchers based on a specific instrument. See How do I select appropriate reporter and quencher combinations for multiplex PCR.

 

FAQ ID -9093
Can I use uracil-N-glycosylase (UNG) with the QuantiFast and Rotor-Gene PCR kits?

No. UNG treatment does not provide any advantage for the QuantiFast and Rotor-Gene PCR kits because the mastermixes do not contain dUTP. Use the QuantiTect kits if you intend to use the UNG treatment.

FAQ ID -9092
Why do I see multiple high-intensity peaks in my qPCR dissociation curve at temperatures less than 70ºC?

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.

 

Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

 

FAQ ID -90990
How do I quantify gene expression levels if the amplification efficiencies are different between the genes of interest and endogenous reference gene?

The REST 2009 (Relative Expression Software Tool) software applies mathematic models that compensate for the different PCR efficiencies of the gene of interest and reference genes. In addition, the software can use multiple reference gene normalization to improve the reliability of result, as well as provides statistical information suitable for robust comparison of expression in groups of treated and untreated. QIAGEN offers the REST 2009 software free of charge.

FAQ ID -9095
What is the threshold cycle or Ct value?
The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.
FAQ ID -2682
What do I do if no fluorescent signal is detected in a real-time PCR assay?

Check the template quality and integrity by amplifying an endogenous control gene. Check the amplicon by QIAxcel Advanced system or agarose gel electrophoresis to show that amplification was successful.

 

Determine whether the gene of interest is expressed in your sample. See How can I find out if my gene of interest is express in a specific tissue type or cell line.  Ensure the assay setup and cycling conditions are correct, and that the data collection channel matches the emission wavelength of the fluorescent dye used. Use a control sample in which the gene of interest is definitely expressed.

 

If the issue persists, please send the original run file to QIAGEN Technical Services.

FAQ ID -9091
How do I select appropriate reporter and quencher combinations for multiplex PCR?

For duplex analysis, using non-fluorescent quenchers (e.g., Black Hole Quencher®) is preferred over fluorescent quenchers (e.g., TAMRA fluorescent dye). For triplex and 4-plex analysis, QIAGEN strongly recommends using non-fluorescent quenchers. Generally, use the green channel, the yellow channel, and the orange and crimson channels to detect the least abundant target, the second least abundant target, and the two most abundant targets, respectively. For instrument-specific recommendations, please see the handbooks for the QuantiTect Multiplex PCR kit, QuantiFast Multiplex kit or Rotor-Gene Multiplex kit.

 

FAQ ID -9094
Can I skip the gDNA wipeout buffer treatment step for the QuantiTect Reverse Transcription Kit?

The gDNA wipeout buffer incubation step can be skipped when the total RNA is free from genomic DNA. However, the gDNA wipeout buffer is still required to be added because the reverse transcription step is optimized in the presence of components in the gDNA wipeout buffer.

FAQ ID -9098
How should I handle and store absolute quantitation standards for real-time experiments?
Store the standards at a high concentration in aliquots at -20oC to -70oC. If using low concentrations, stabilize standards with carrier nucleic acid. It is always best to use freshly diluted standards for each experiment. If possible, use siliconized tubes for standard (and target) dilutions. This will prevent any unspecific binding of nucleic acids to the plastic.
FAQ ID -9099
How do I ensure reliable results for High Resolution Melting (HRM) assays?

Reliable HRM analysis results depend on template quality, highly specific HRM PCR kit with a saturation dye, a real-time instrument with HRM capability, and powerful software package. Factors critical for successful HRM analysis are:

 

  • Use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids variation due to differing composition of elution buffers.
  • DNA template concentrations should be normalized using the same dilution buffer. Ensure the CT values are below 30 and differ no more than 3 CT values across individual samples.
  • Design assays with amplicon length 70–350 bp. For SNP analysis, use amplicon length 70–150 bp.
  • Always start with 0.7 µM primer concentration

 

For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.

FAQ ID -9097
Why should DNA or cDNA targets be less than 250 bp long for real-time PCR?

Shorter amplification products facilitate high PCR efficiencies. Ideally, amplicon length should be less than 150 bp for optimal amplification efficiency. PCR efficiencies close to 100% are a crucial prerequisite for accurate quantification of target copy numbers in real-time PCR.

FAQ ID-751
What is the difference between QuantiTect Virus Kit and the new QuantiFast Pathogen +IC Kits?
The QuantiTect Virus Kit and the new QuantiFast Pathogen +IC Kits are based on similar reaction chemistry and show identical sensitivity but as a new feature, the QuantiFast Pathogen kits include an Internal Control template and the corresponding primer/probe set for duplex amplification of a user-defined pathogen target with the provided Internal Control. Additional new features are pack size, kit variants, ROX variants, and speed.
FAQ ID -2448
Can the Internal Controls be ordered separately in the QuantiFast Pathogen + IC kit for use during purification?

Yes, the Internal Control RNA (High conc.) and Internal Control DNA (High conc.) templates are available under a separate catalog number. After reconstitution according to the description in the handbook, these IC templates have a 10x higher concentration than the Internal Control templates provided in the kits.   They are sufficiently concentrated to be spiked into the sample prep without replacing too much of the sample input volume.

 

**Please note that there is no assay (primer/probe set) for amplification of the IC included in these separate catalog numbers. The assay is only included in the QuantiFast Pathogen PCR +IC Kit and QuantiFast Pathogen RT-PCR +IC Kit.  The ICs cannot be used with the QuantiTect Virus kits because the assay (primer/probe set) for the detection of the IC is provided with the QuantiFast Pathogen Kits only.

 

FAQ ID -2451
How do I avoid collecting a fluorescence reading from primer-dimer with the QuantiTect SYBR Green PCR Kit?

Depending on primer design and copy number of target, primer-dimer may occur and its signal might be detected. Typical strategies against this are to optimize PCR conditions and/or redesign the assay.

 

Alternatively, an additional data-acquisition step can be added to the 3-step cycling protocol. First, determine the melting temperatures (Tm) for both the amplicon and the primer-dimer. Then, add a 15 second data-acquisition step with a temperature that is higher than the primer-dimer Tm, but approximately 3ºC lower than the specific amplicon Tm.

FAQ ID -9096