QuantiTect Virus Kit

适用于高灵敏度检测病毒 RNA 和/或 DNA

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QuantiTect Virus Kit (1000)

Cat. No. / ID: 211015

适用于 1000 次 50 µl 反应：QuantiTect Virus Master Mix（含 ROX 染料）、QuantiTect Virus RT Mix、RNase-Free Water、QuantiTect Nucleic Acid Dilution Buffer

Reference dye

Rox in Mastermix

Rox in vial

Reactions

1000

200

Inquire

QuantiTect Virus Kit 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。

✓ 全天候自动处理在线订单

✓ 博学专业的产品和技术支持

✓ 快速可靠的（再）订购

特点

高灵敏度的单次和多重检测
在同一反应中检测病毒 RNA 和/或 DNA
明确地检测微弱的阳性信号
快速的通用 2 步方案
5 倍预混液，用于在增加样本输入的情况下提高灵敏度

产品详情

QuantiTect Virus Kit 专门设计用于使用序列特异性探针，通过多重、实时一步法 RT-PCR 高灵敏度地检测多达 4 个病毒核酸靶标。可在单个试管中检测多个病毒 RNA 和/或 DNA 靶标以及内部对照品而不会降低灵敏度。浓缩的 5 倍预混液可添加更高的样品输入，并含有 HotStarTaq Plus DNA Polymerase。QuantiTect Virus RT Mix 含有 Sensiscript Reverse Transcriptase 的独特配方，针对高灵敏度病毒 RNA 检测进行了优化。有两种试剂盒格式可供选择：适用于需要 ROX 染料进行荧光归一化处理的循环仪的 QuantiTect Virus Kit；适用于其他循环仪的 QuantiTect Virus +ROX Vial Kit。为方便起见，预混液可存放在 2-8°C 环境下。

绩效

使用 QuantiTect Virus Kit 进行扩增时，即使模板量低而 C_T 值高，也能在一定稀释度范围内呈现陡峭的 S 形曲线（请参阅图“ 在宽动态范围内明确测定 C_T 值”）。这样就能在 Real-time PCR 中准确测定病毒核酸定量的 C_T 值。

多重检测可在宽线性范围内检测多个病毒 RNA 和/或 DNA 靶标及内部对照品而不会降低灵敏度（请参阅图“ 在宽线性范围内可靠地检测病毒 RNA”和“ 改善了低量病毒 RNA 的检测”）。

试剂盒附带的 QuantiTect Nucleic Acid Dilution Buffer 可在稀释和反应设置过程中稳定 RNA 和 DNA 标准品，并防止试管或移液器吸头等塑料表面的核酸流失。该缓冲液可对用于病毒核酸定量的标准品进行可靠的稀释，从而提供从低到高 C_T 值的宽线性范围，并确保标准品长期存放而不会发生降解（请参阅图“ 可靠的 RNA 标准品稀释和存放”）。

查看图表

原理

QuantiTect Virus Kit 可在单次或多重检测中首次尝试即实现高灵敏度的病毒核酸检测（请参阅流程图“ QIAGEN 多重试剂盒”）。经优化的预混液可确保多重反应中 PCR 产物的扩增效率和灵敏度与相应单次扩增反应中的 PCR 产物相同。

在同一而非独立反应中扩增对照品和靶标基因可尽量减少操作错误，从而提高基因定量的可靠性。QuantiTect Virus Buffer 包含 K⁺ 和 NH₄⁺ 离子的均衡组合以及独特的 Synthetic Factor MP，它们能共同促进引物和探针稳定且高效地退火到核酸模板，从而提高 PCR 的效率（请参阅图“ 独特的 PCR 缓冲液”）。此外，Sensiscript Reverse Transcriptase 的独特配方可确保病毒 RNA 的高灵敏度逆转录，而 HotStarTaq Plus DNA Polymerase 可提供严格的热启动，防止形成非特异性产物。

QuantiTect Virus Kit 的组分
	试剂盒组成	特点	优势
5x QuantiTect Virus Master Mix	浓缩预混液	高度浓缩，专为灵敏的病毒检测而优化	可在检测中加入更大体积的模板以提高灵敏度
	HotStarTaq Plus DNA Polymerase	在 95ºC 下 5 分钟活化	在室温下设置 qPCR 反应
	QuantiTect Virus Buffer	NH₄⁺ 和 K⁺ 离子的平衡组合	特异性引物退火确保可靠的 PCR 结果
	QuantiTect Virus Buffer	Synthetic Factor MP	可在同一试管中对多达 4 个基因进行可靠的多重分析
其他试剂盒组分	QuantiTect Virus RT Mix	含有 Sensiscript Reverse Transcriptase 的独特配方	针对病毒 RNA 的高灵敏度检测进行了优化
其他试剂盒组分	QuantiTect 核酸稀释缓冲液	用于稀释和存放核酸标准品的专有缓冲液制剂。	在稀释和反应设置过程中稳定 RNA 和 DNA 标准品，并防止试管或移液器吸头等塑料表面的核酸流失

查看图表

QIAGEN 多重试剂盒。

独特的 PCR 缓冲液。

程序

QuantiTect Virus Kit 使用序列特异性探针对病毒核酸（RNA 和/或 DNA）和内部对照品进行高灵敏度的 Real-time PCR 分析。反应可在包含或不包含逆转录步骤的情况下进行，从而可灵活设计多重检测来检测 RNA 靶标、DNA 靶标或同时检测 RNA 和 DNA 靶标。请按照手册中的方案来获得快速可靠的结果。

试剂盒提供时预混液中可包含或不含 ROX 参比荧光染料（见表）。

选择合适的 QuantiTect Virus Kit
ROX 染料	试剂盒	兼容循环仪
在预混液中提供	QuantiTect Virus Kit	Applied Biosystems 除 Applied Biosystems 7500 外的所有循环仪
以单独试管提供	QuantiTect Virus +ROX Vial Kit	Applied Biosystems 7500 和来自 Bio-Rad、Cepheid、Eppendorf、QIAGEN、Roche、Agilent 及其他供应商的循环仪

对于快速、高灵敏度的终点一步法 RT-PCR 应用（包括病毒检测），我们建议使用 QIAGEN OneStep RT-PCR Kit。

应用

QuantiTect Virus Kit 使用序列特异性探针提供高灵敏度的实时单次或多重 PCR 或一步法 RT-PCR，用于检测病毒 DNA 和/或 RNA 以及内部对照品。这些试剂盒可用于多种实时热循环仪，包括来自 Applied Biosystems、Bio-Rad、Cepheid、Eppendorf、QIAGEN、Roche（毛细管循环仪除外）和 Agilent 的仪器。

辅助数据和图表

在宽动态范围内明确测定 C_T 值。

使用 QuantiTect Virus Kit 扩增并分析了所示的牛病毒性腹泻病毒 (Bovine Viral Diarrhea Virus, BVDV) RNA 的连续稀释液。曲线呈 S 形，即使在模板量较低（10 个拷贝/μl）的情况下也能准确测定 C_T 值。[A] 检测 BVDV1 RNA。[B] 检测 BVDV2 RNA。NTC：无模板对照品。

Specifications

Features	Specifications
Applications	病毒检测
SYBR Green I or sequence-specific probes	序列特异性探针
Real-time or endpoint	实时
Reaction type	逆转录和 PCR
Thermal cycler	大多数实时循环仪（毛细管循环仪除外，例如 LightCycler® 1.x 和 2.0）
Sample/target type	RNA 和/或 DNA 靶标
With or without ROX	可以预混液中 ROX 和独立小瓶 ROX 形式提供
Single or multiplex	单次或多重

资源

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

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