QuantiFast Pathogen +IC Kit

用于灵敏而又可靠地检测病毒 RNA/DNA 和细菌 DNA，包括内质控

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QuantiFast Pathogen RT-PCR +IC Kit (400)

Cat. No. / ID: 211454

适用于 400 次 25 µl 反应：Master Mix、RT Mix、Lyophilized Internal Control Assay、Lyophilized Internal Control RNA、ROX Dye Solution、High-ROX Dye Solution、RNase-Free Water、Nucleic Acid Dilution Buffer、Buffer TE

KitControl

QuantiFast Pathogen Kit

Internal Control

Type

RT-PCR

PCR

Inquire

见商标。

QuantiFast Pathogen +IC 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

同时检测病原体靶标和内质控
5x 浓度预混液，用于通过提高样品的投入量而提高灵敏度
正确解读阴性结果，提高过程安全性
明确地检测微弱的阳性靶标信号
标准和快速定量 PCR 仪上的快速通用方案

产品详情

QuantiFast Pathogen +IC Kit 设计用于使用序列特异性探针对病原体核酸进行灵敏而又快速的 Real-time PCR 或一步法 RT-PCR 检测。为了能够通过正确解读阴性检测结果实现高过程安全性，每个试剂盒都包含用于多重实时检测多达 4 种目标病原体靶标（例如病毒、细菌、真菌等）的试剂以及内质控 (Internal Control, IC)。有两种试剂盒形式可供选择：用于检测病毒 RNA 的 QuantiFast Pathogen RT-PCR +IC Kit，其中包括 RNA 内质控模板，以及内质控引物/探针组合；或用于检测病毒、细菌或真菌 DNA 的 QuantiFast Pathogen PCR +IC Kit，其中包含 DNA 内质控模板，以及内质控引物/探针组合。在这两种试剂盒中，都提供有 2 管具有不同浓度的 ROX，使得可以在几乎任何实时定量 PCR 仪器上使用。为方便起见，预混液可存放在 2-8°C 环境下。

绩效

QuantiFast Pathogen +IC Kit 可在宽线性范围内同时检测病毒 RNA 或 DNA 靶标及所提供的内质控，且在多重检测时不会降低灵敏度（请参阅图在 Rotor-Gene Q 上进行的诺如病毒和单重和双重检测具有高线性度和精度）。该方案的开发宗旨是在大多数定量 PCR 仪上进行快速、可靠性高的循环反应实验（请参阅图“ 在 Rotor-Gene Q 上灵敏检测 BHV-1”和“ 在 ABI 7500 上灵敏检测 BHV-1”）。使用 QuantiFast Pathogen +IC Kit 对病原体靶标和内质控同时进行扩增，可确保正确解读阴性结果，从而提高病原体检测工作流程的安全性（请参阅图“ 阴性结果的正确解读”）。

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，并防止反应管或移液器吸头等塑料表面的核酸残留。它能可靠地稀释用于病毒核酸定量的标准品，从而提供从低到高 C_T 值的宽线性范围。缓冲液可确保标准品长期保存而不发生降解（请参阅图“ 可靠的 RNA 标准品稀释和保存”）。

查看图表

灵敏检测诺如病毒。

单重和双重检测具有高线性度和精度。

在 Rotor-Gene Q 上灵敏检测 BHV-1。

在 ABI 7500 上灵敏检测 BHV-1。

阴性结果的正确解读。

可靠的 RNA 标准品稀释和保存。

原理

为了能够通过正确解读阴性检测结果实现高过程安全性，每个 QuantiTect Pathogen _IC Kit 都包含用于多重实时检测用户目标病原体靶标的试剂以及内质控。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。

QuantiFast Pathogen +IC Kit 可在首次尝试时便能成功对病原体核酸进行灵敏、快速的实时多重 PCR 或一步法 RT-PCR 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重扩增反应中的 PCR 产物相同。专门开发的快速 PCR 缓冲液含有新型添加剂 Q-Bond，可显著缩短变性、退火和延伸时间（请参阅图“ 快速引物退火”）。K⁺ 和 NH₄⁺ 离子的均衡组合以及独特的 Synthetic Factor MP 能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。

QuantiFast Pathogen +IC Kit 的组分
	试剂盒组成	特点	优势
5x QuantiFast Pathogen PCR Master Mix	浓缩预混液	高度浓缩，专为灵敏的病原体检测而优化	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	在室温下配制 qPCR 反应
	QuantiFast Pathogen 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
		Synthetic Factor MP	可在一管反应中对多达 4 个基因进行可靠的多重分析
		独特的 Q-Bond 添加剂	更快的 PCR 运行时间，从而提高每天的结果产生速度和反应次数
Internal Control Assay	内质控模板	QuantiFast Pathogen PCR +IC Kit 中的内质控 DNA 模板	一种通用的 DNA 扩增对照品，可用于不同的病原体检测
	内质控模板	QuantiFast Pathogen RT-PCR +IC Kit 中的 Internal Control DNA	一种通用的 RNA 扩增对照品，可用于不同的病原体检测
	Internal Control Assay	用 MAX（相当于 HEX、VIC 等）标记的预混合引物/探针组合（TaqMan® 探针）	不会干扰针对病原体-靶标的引物
其他试剂盒组分	QuantiFast Pathogen RT Mix*	含有 Sensiscript Reverse Transcriptase 的独特配方	针对病原体 RNA 的高灵敏度检测进行了优化
	ROX Dye Solution	单独的参比荧光染料管，用于在 Applied Biosystems 7500 Real-Time PCR System 上对荧光信号进行归一化处理。可选：可用于 Agilent 的 Stratagene 仪器	可在需要 ROX 染料的定量 PCR 仪上精确定量。在任何实时定量 PCR 仪上都不会干扰 PCR
	High-ROX Dye Solution	单独的参比荧光染料管，用于在 Applied Biosystems 7900 和 StepOne Real-Time PCR System 上对荧光信号进行归一化处理	可在需要 ROX 染料的定量 PCR 仪上精确定量。在任何实时定量 PCR 仪上都不会干扰 PCR
	QuantiTect 核酸稀释缓冲液	用于稀释和保存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，并防止反应管或移液器吸头等塑料表面的核酸残留

* 仅包含在 QuantiFast Pathogen RT-PCR +IC Kit 中

查看图表

QIAGEN 多重试剂盒。

快速引物退火。

独特的 PCR 缓冲液。

程序

QuantiFast Pathogen +IC Kit 提供了一种检测用户目标病原体和内质控的简单程序。它们含有即用型预混液，用于实时检测病毒 RNA（1 步法 RT-PCR）或病毒、细菌和真菌 DNA (PCR)。无需优化反应和循环反应条件。只需将提供的预混液与病原体检测（引物和探针）以及提供的 Internal Control Assay 和 Internal Control DNA 或 RNA 混合。或者，如果在样本纯化过程中已经加入了内质控，则在反应混合物中加入无 RNase 水来替代 Internal Control DNA 或 RNA。然后加入样品 DNA 或 RNA，并在任意定量 PCR 仪上启动反应。试剂盒手册中包含在各种定量 PCR 仪上与 TaqMan® 探针一起使用的经优化方案。其中还包含染料组合的选择建议。

每个 QuantiFast Pathogen +IC Kit 都包含 Internal Control Assay 和 Internal Control DNA 或 RNA，可直接加入反应混合物中作为扩增对照品使用。或者，也可将 IC 添加到纯化程序中，以对纯化过程和扩增进行质控。如需在纯化过程中添加内质控，可单独订购高浓度 Internal Control DNA 或 RNA (High conc.)（请参阅图“ QIAGEN Internal Control”）。

查看图表

QIAGEN Internal Control。

应用

QuantiFast Pathogen +IC Kit 使用序列特异性探针进行灵敏的 Real-time PCR 或一步法 RT-PCR，用于检测包含了内质控的病原体 DNA 、RNA。这些试剂盒可用于多种实时定量 PCR 仪，包括 QIAGEN、Applied Biosystems、Bio-Rad、Roche（毛细管循环仪除外）和 Agilent 的定量 PCR 仪。

辅助数据和图表

阴性结果的正确解读。

在存在不同量的 PCR 抑制物质（腐植酸）的情况下，在 Rotor-Gene Q 上将两种浓度的病毒 RNA 靶标与内质控进行了双重复共扩增。[A] 无模板对照 (No template control, NTC) 作为内质控信号的参照。[B] 病毒 RNA 靶标和内质控的扩增表明成功进行了扩增。[C] 内质控表明存在少量抑制剂。[D] 检测不到内质控表明扩增反应因抑制剂的存在而失败。

Specifications

Features	Specifications
Applications	病原体检测：病毒、细菌或真菌 DNA 的 Real-time PCR (QuantiFast Pathogen PCR +IC Kit) 或检测病毒 RNA 的一步法 RT-PCR (QuantiFast Pathogen RT-PCR +IC Kit)
Sample/target type	QuantiFast Pathogen PCR +IC Kit：病毒、细菌或真菌 DNA；QuantiFast Pathogen RT-PCR +IC Kit：病毒 RNA
Single or multiplex	双重
Reaction type	Real-time PCR 或一步法 RT-PCR，包括内质控 (Internal Control, IC)
Real-time or endpoint	实时
SYBR Green I or sequence-specific probes	序列特异性探针
Thermal cycler	适用于大多数与双重 PCR/RT-PCR 兼容的标准和快速实时定量 PCR 仪，例如 Rotor-Gene Q 或 Agilent、Applied Biosystems、BioRad 和 Roche 的定量 PCR 仪
With or without ROX	预混液以不含 ROX 染料的形式提供，但包括了 2 管独立包装的 ROX 溶液：用于除 ABI 7500 以外的 ABI PCR 仪的 High-ROX Dye Solution、用于 ABI 7500 和其他供应商定量 PCR 仪的 ROX Dye Solution（低浓度 ROX）

资源

配列特異的なプローブを用いた高感度な1 ステップリアルタイムRT-PCR によるウイルスRNA およびインターナルコントロールの検出

配列特異的なプローブを用いた高感度リアルタイムPCR によるウイルス／バクテリアDNA およびインターナルコントロールの検出

Download Safety Data Sheets for QIAGEN product components.

Now with even more applications!

FAQ

The detection limit for the QuantiFast Pathogen RT-PCR and PCR kits is < 10 copies.

FAQ ID -2453

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

The DNA IC is a non-linearized plasmid. The RNA IC is an in vitro transcript. Both are naked nucleic acids. The size (base pair length) of both templates is sufficient to allow for efficient purification with standard methods for nucleic acid extraction.

FAQ ID -2450

Replicates in real-time PCR may have different plateau heights due to differences in the reaction kinetics for each sample. Even though replicates start out with identical template amounts, the rate at which reagents are being depleted, and the point when exponential accumulation of PCR product stops and becomes linear, differ between replicates. This will result in different plateau heights, the stage where PCR reactions have come to a halt, and little or no additional PCR product is being amplified. You can find further information in Chapter 'Quantification of target amounts' of our Brochure "Critical Factors for Successful Real-Time PCR".

FAQ ID -539

The QuantiFast Pathogen PCR +IC Kit and the QuantiFast Pathogen RT-PCR +IC Kit are supplied with master mixes that do not contain ROX. Instead, 2 different ROX concentrations are supplied in separate tubes. “High-ROX Dye Solution” is suitable for use with all ABI cyclers except ABI 7500, and “ROX Dye Solution” is suitable for use with ABI 7500 and, optionally, instruments from Stratagene (Agilent). Recommendations are provided in the handbook.

FAQ ID -2605

Using the QuantiFast Pathogen + IC kits on the Rotor-Gene Q:

RT-PCR
40 cycles	~95 minutes
45 cycles	~100 minutes

PCR
40 cycles	~75 minutes
45 cycles	~80 minutes

FAQ ID -2452

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

The QuantiFast Pathogen PCR + RT-PCR +IC Kits can be used on all leading block cycler platforms, but not on capillary systems (e.g., LightCycler 2.0).

FAQ ID -2596

No calibration for MAX is needed if the instrument is calibrated for VIC. Use the FAM/SYBR Green filter for the pathogen assay and the VIC/JOE filter for the IC assay. To detect the Internal Control (MAX) in the VIC/JOE filter, create a new detector (e.g., “MAX/IowaBlack”). Assign “VIC” as the reporter dye and “None” for the quencher dye.

FAQ ID -2610

Please follow the instructions in the handbook. Briefly, use gain optimization “Before First Acquisition” for the pathogen assay (FAM) in the Green channel, and use a fixed gain of 9 for the Internal Control Assay (MAX) in the Yellow channel.

FAQ ID -2608

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

MAX can be detected using the same channels or filters as for HEX, JOE, or VIC.

FAQ ID -2597

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

No, the Internal Controls used in the QuantiFast Pathogen PCR +IC Kit and the QuantiFast Pathogen RT-PCR +IC Kit are an artificial sequence that is not present in biological sample material.

FAQ ID -2598

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

No, the QuantiFast Pathogen PCR +IC kits are designed for use with hydrolysis probes (also known as TaqMan® probes) only.

See trademarks.

FAQ ID -2595

Typically, the target-specific probe should be labeled with FAM as the reporter dye and a non-fluorescent quencher (e.g., Dark Quencher, Black Hole Quencher [BHQ] or Iowa Black Quencher). Other reporter dyes than FAM, detected in a different detection channel than MAX, may also be suitable. It is not recommended to use fluorescent quenchers (e.g., TAMRA fluorescent dye). Due to their own native fluorescence, fluorescent quenchers contribute to an overall increase in background and reduce the signal-to-noise ratio.

FAQ ID -2594

No, the Internal Control template DNA or RNA must be added to the lysis buffer or to the lysate in order to prevent the loss of Internal Control template thorough matrix effects.

FAQ ID -2602

In the “Filter Gain Settings” dialog box, set the filter gain to a value of 4 for both the FAM/SYBR Green and HEX/JOE/VIC filters. See the Mx instrument/software manual for details.

FAQ ID -2609

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

Make sure that template, primers, probes, and amplification reagents are stored correctly and avoid multiple freeze–thaw cycles for oligonucleotides and template. Check the performance of your real-time instrument as some instruments require the halogen lamp to be frequently replaced. Lasers must also be replaced occasionally.

FAQ ID -589

Both membrane and magnetic bead-based kits have been tested (e.g., QIAamp MinElute Virus Kit, QIAamp Viral RNA Kit, DNeasy kits, RNeasy kits, QIAsymphony Virus/Bacteria kits, QIAxtractor Vx Reagents).

FAQ ID -2606

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

Principally, yes. However, additional factors influencing the CT are:

1. Extraction efficiency, which depends on extraction method and sample material.

2. Volume of the eluate used for PCR.

Examples: Use of the Internal Control according to handbook instructions (0.1µl per 1µl elution volume) for two different workflows.

Workflow 1: High extraction efficiency: use of 5 µl of the eluate for PCR results in a CT value of 29-30.
Workflow 2: High extraction efficiency: use of 10 µl of the eluate for PCR results in a CT of 28-29.

Depending on the workflow, the amount of Internal Control to be added to the extraction might have to be adjusted to more than 0.1 µl per 1 µl elution volume, or to less than 0.1 µl per 1 µl elution volume, in order to achieve a CT value within the expected range.

Running the Internal Control DNA/RNA provided with the QuantiFast Pathogen +IC Kit in a separate reaction can serve as a reference for adjusting the amount of Internal Control DNA/RNA (High conc.) to be added to the extraction.

FAQ ID -2604

On the Rotor-Gene Q, setting the threshold in the Yellow channel to an absolute value of 0.05 will give satisfactory results in most cases.

FAQ ID -2607

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

In the QuantiFast Pathogen + IC kit, the probe is labeled with MAX-NHS ester (MAX) as the reporter dye which has a spectrum equivalent to HEX, JOE or VIC dyes. IowaBlack is used as the quencher dye. IowaBlack is a non-fluorescent quencher (dark quencher).

FAQ ID -2449

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

After reconstitution according to handbook instructions, the Internal Control (IC) templates provided with the kits have a ready-to-use concentration for direct use as an amplification control in PCR or RT-PCR. For each 25 µl reaction, 2.5 µl of the IC are added, resulting in an expected CT value of approximately 29-30.

In order to achieve the same CT value when using the IC as an extraction control, more IC template has to be spiked in before extraction. The exact amount depends mainly on the elution volume. For each 1 µl of elution volume, 0.1 µl of the IC High conc. should be added to each of the extraction samples. This should result in a CT value in the PCR or RT-PCR reactions of approximately 29-30.

Examples: For 100 µl of elution volume, 10 µl of the IC, per sample, should be added to the lysis buffer or lysate. For 50 µl of elution volume, 5 µl of the IC, per sample, should be added to the lysis buffer or lysate.

FAQ ID -2603

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

The Internal Control templates can be freeze-thawed for up to 6 times without decrease in performance. However, great care should be taken to avoid inadvertently introducing RNAses/DNAses into the Internal Control template solutions. In general, in order to avoid repeated freeze-thaw cycles, we recommend preparing aliquots of the Internal Control templates.

FAQ ID -2599

When the IC is added to the amplification reaction mix, the IC signal confirms that PCR amplification has been successful. When it is added at the extraction step to the lysis buffer or lysate, the IC signal confirms that nucleic acid purification and PCR amplification have been successful.

FAQ ID -2600

Yes, the Internal Control RNA will only give the expected signal when both the reverse transcription and the PCR reactions have been successful.

FAQ ID -2601