QuantiTect Virus Kits

高灵敏度检测病毒RNA或DNA

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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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Reference dye

Rox in Mastermix

Rox in vial

Reactions

1000

200

QuantiTect Virus Kits 旨在用于分子生物学应用。这些产品不能用于疾病诊断、预防和治疗。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

高灵敏度的单一和多重检测
在同一反应中检测病毒RNA和/或DNA
清晰检测弱阳性信号
通用的快速两步法实验方案
5x预混液灵敏度更高，起始样本量更大

Product Details

QuantiTect Virus Kit专用于病毒核酸的高灵敏检测，使用序列特异性的探针，通过多重一步法real-time RT-PCR可对多至4个病毒核酸靶标进行检测。多重分析可对含有内参的多种病毒RNA和/或DNA靶分子进行检测，不会影响灵敏度。试剂盒中浓缩的5x预混液可实现更大上样量，还包含HotStarTaq Plus DNA Polymerase。QuantiTect Virus RT Mix含有独特设计的Sensiscript Reverse Transcriptase，经过优化可进行病毒RNA的高灵敏度检测。该试剂盒有两种规格：QuantiTect Virus Kit适用于需要ROX染料进行荧光校正的PCR仪，QuantiTect Virus +ROX Vial Kit适用于其他PCR仪。为便于使用，预混液可储存在2–8°C。

Performance

应用QuantiTect Virus Kits进行扩增可在广泛的稀释范围内获得陡峭的Sigma曲线，即使在模板量少、C_T值高的情况下也是如此（参见" Unambiguous determination of C_T values over a wide dynamic range"）。由此可精确确定real-time PCR的C_T值，定量分析病毒核酸。

多重分析可检测含有内参的多种病毒RNA和/或DNA靶分子，适用线性范围广泛，且不影响检测灵敏度（参见" Reliable detection of viral RNA over a wide linear range"和" Improved detection of low amounts of viral RNA"）。

试剂盒中提供的QuantiTect Nucleic Acid Dilution Buffer可在稀释和反应体系构建过程中稳定RNA和DNA标准品，避免在试管和枪头等管壁丢失核酸。该缓冲液确保可靠稀释用于病毒核酸定量分析的标准核酸，可获得从低到高C_T值的广泛线性范围。缓冲液可避免降解，延长标准品的储存时间（参见" Reliable dilution and storage of RNA standards"）。

See figures

Unambiguous determination of C_T values over a wide dynamic range.

Reliable detection of viral RNA over a wide linear range.

Improved detection of low amounts of viral RNA.

Reliable dilution and storage of RNA standards.

Principle

QuantiTect Virus Kit可通过单一或多重分析对病毒核酸高度灵敏的检测（参见" QIAGEN multiplex kits"）。优化的预混液保证PCR产物在多重反应中的扩增效率和灵敏度与在单一反应中相同。

在同一反应液中扩增内参和靶基因，可减少手工误差，提高基因定量分析的可靠性。QuantiTect Virus Buffer含有浓度平衡的K⁺、NH₄⁺和独特合成的MP因子，可促使引物和探针与核酸模板稳定、高效的退火，提高PCR效率（参见" Unique PCR buffer"）。此外，Sensiscript Reverse Transcriptase确保对病毒RNA高灵敏度的逆转录，而HotStarTaq Plus DNA Polymerase具有严格的热启动，可防止非特异性产物的形成。

QuantiTect Virus Kit的组份
	试剂盒组份	特点	优势
5x QuantiTect Virus Master Mix	浓缩的预混液	浓度高，经过优化，可灵敏的检测病毒核酸	起始样本量更大，灵敏度更高
	HotStarTaq Plus DNA Polymerase	在95ºC下5分钟即可活化	室温下构建qPCR反应体系
	QuantiTect Virus Buffer	NH₄⁺和K⁺的平衡组合	特异性引物退火确保PCR结果可靠
	QuantiTect Virus Buffer	合成的Factor MP	在单管内对4个基因进行可靠的多重分析
其他的试剂盒组份	QuantiTect Virus RT Mix	Contains a unique formulation of Sensiscript Reverse Transcriptase	优化用于高度灵敏检测病毒RNA
其他的试剂盒组份	QuantiTect Nucleic Acid Dilution Buffer	Proprietary buffer formulation for dilution and storage of nucleic acid standards.	在稀释和反应体系构建时稳定RNA和DNA标准，避免核酸在塑料表面（如指管或枪头）的损失

See figures

QIAGEN multiplex kits.

Unique PCR buffer.

Procedure

QuantiTect Virus Kit使用序列特异性探针可对病毒核酸（RNA和/或DNA）及内参进行高度灵敏的real-time PCR分析。反应可选择是否进行逆转录步骤，可灵活设计实验检测RNA、DNA或者RNA及DNA。选用使用手册中的实验方案能快速获得可靠结果。

可选预混液中含有或不含ROX染料的试剂盒（见下表）。

选择合适的QuantiTect Virus Kit
ROX染料	试剂盒	兼容的PCR仪
预混液中含有ROX染料	QuantiTect Virus Kit	除Applied Biosystems 7500以外的Applied Biosystems的各种PCR仪
预混液中不含ROX染料	QuantiTect Virus +ROX Vial Kit	Applied Biosystems 7500和 Bio-Rad、Cepheid、 Eppendorf、QIAGEN, Roche、Agilent等PCR仪

我们推荐使用QIAGEN OneStep RT-PCR Kit进行快速、高灵敏度的终点式一步法RT-PCR，包括病毒检测。

Applications

QuantiTect Virus Kit可进行高度灵敏的单一或多重real-time PCR，或应用序列特异性探针的一步法RT-PCR，检测内参和病毒DNA和/或RNA。该试剂盒可用于各种real-time PCR仪，包括Applied Biosystems、Bio-Rad、Cepheid、Eppendorf、QIAGEN、Roche（除capillary cyclers以外）和Agilent的PCR仪。

Supporting data and figures

Unambiguous determination of C_T 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 C_T value determination, even at low template amounts (10 copies/μl). [A] Detection of BVDV1 RNA. [B] Detection of BVDV2 RNA. NTC: No template control.

Specifications

Features	Specifications
Applications	Virus detection
SYBR Green I or sequence-specific probes	Sequence-specific probes
Real-time or endpoint	Real-Time
Reaction type	Reverse Transcription and PCR
Thermal cycler	Most real-time cyclers (except capillary cyclers e.g. LightCycler® 1.x and 2.0)
Sample/target type	RNA and/or DNA targets
With or without ROX	Available with ROX in master mix and ROX as a separate vial
Single or multiplex	Single or multiplex

Resources

Now with even more applications!

Download Safety Data Sheets for QIAGEN product components.

For highly sensitive real-time singleplex or multiplex PCR or one-step RT-PCR using sequence-specific probes for detection of viral DNA and RNA and internal controls

Now with even more applications!

For highly sensitive real-time singleplex or multiplex PCR or one-step RT-PCR using sequence-specific probes for detection of viral DNA and RNA and internal controls

FAQ

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

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

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

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

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

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

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

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

Store the standards at a high concentration in aliquots at -20^oC to -70^oC. 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

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 C_T values are below 30 and differ no more than 3 C_T 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

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

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

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

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 (T_m) 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 T_m, but approximately 3ºC lower than the specific amplicon T_m.

FAQ ID -9096

QuantiTect Virus Kits

高灵敏度检测病毒RNA或DNA

Features

Product Details

Performance

See figures

Principle

See figures

Procedure

Applications

Supporting data and figures

Unambiguous determination of CT values over a wide dynamic range.

Specifications

Resources

FAQ

Unambiguous determination of C_T values over a wide dynamic range.