QuantiFast Pathogen +IC Kits
For sensitive and reliable detection of viral RNA/DNA and bacterial DNA, including internal control
- Simultaneous detection of pathogen target and internal control
- 5x master mix for higher sensitivity with more sample input
- Correct interpretation of negative results for increased process safety
- Clear detection of weak positive target signals
- Fast, universal protocol on both standard and fast cyclers
The QuantiFast Pathogen +IC Kits are designed for sensitive and rapid real-time PCR or one-step RT-PCR detection of pathogen nucleic acids using sequence-specific probes. To enable high process safety through correct interpretation of negative detection results, each kit contains reagents for multiplex real-time detection of up to 4 user-defined pathogen targets (e.g., virus, bacteria, fungi etc.) plus the Internal Control (IC). Two kit formats are available: The QuantiFast Pathogen RT-PCR +IC Kit for detection of viral RNA, which contains an internal RNA control template, plus the Internal Control primer/probe set, or the QuantiFast Pathogen PCR +IC Kit for detection of viral, bacterial, or fungal DNA, which contains an internal DNA control template, plus the Internal Control primer/probe set. With both kits, ROX is supplied in 2 tubes of different concentrations, enabling use on virtually any real-time instrument. For convenience, the master mix can be stored at 2–8°C.
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QuantiFast Pathogen PCR +IC Kit (100)
For 100 x 25 µl reactions: Master Mix, lyophilized Internal Control Assay, lyophilized Internal Control DNA, ROX Dye Solution, High-ROX Dye Solution, RNase-Free Water, Nucleic Acid Dilution Buffer, Buffer TE
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211352
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QuantiFast Pathogen PCR +IC Kit (400)
For 400 x 25 µl reactions: Master Mix, lyophilized Internal Control Assay, lyophilized Internal Control DNA, ROX Dye Solution, High-ROX Dye Solution, RNase-Free Water, Nucleic Acid Dilution Buffer, Buffer TE
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211354
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QuantiFast Pathogen RT-PCR +IC Kit (400)
For 400 x 25 µl reactions: 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
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211454
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Internal Control DNA (High conc.)
For approximately 200 preps (depending on the elution volume): Lyophilized Internal Control DNA, Nucleic Acid Dilution Buffer
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211392
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Internal Control RNA (High conc.)
For approximately 200 preps (depending on elution volume): Lyophilized Internal Control RNA, Nucleic Acid Dilution Buffer
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211492
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QuantiFast Pathogen +IC Kits适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。
See trademarks.
Correct interpretation of negative results.|Sensitive detection of BHV-1 on the Rotor-Gene Q.|Sensitive detection of Norovirus.|Sensitive detection of BHV-1 on the ABI 7500.|Fast primer annealing.|QIAGEN multiplex kits.|Unique PCR buffer.|QIAGEN Internal Control.|High linearity and precision of singleplex and duplex detection.|Reliable dilution and storage of RNA standards.|
Duplicates of two concentrations of a viral RNA target were co-amplified with the Internal Control in the presence of different amounts of a PCR inhibitory substance (humic acid) on the Rotor-Gene Q. [A] No template controls (NTCs) serve as a reference for Internal Control signal. [B] Amplification of viral RNA target and Internal Control confirms successful amplification. [C] The Internal Control indicates the presence of a low amount of inhibitors. [D] Failure to detect the Internal Control shows the failure of the amplification reaction through presence of inhibitors.|Bovine herpes virus type 1 was serially diluted (100 to 10-4) and detected by either singleplex real-time PCR or by duplex real-time PCR in parallel with the Internal Control. Real-time PCR was carried out using the QuantiFast Pathogen PCR +IC Kit on the Rotor-Gene Q according to the supplied protocols and without any PCR optimization. The duplex reactions contained a fixed amount of Internal Control template. Each dilution was analyzed in triplicate; one replicate is shown for each dilution.|A Norovirus RNA transcript was serially diluted (100 to 10-5) and detected by either singleplex real-time RT-PCR or by duplex real-time PCR in parallel with the Internal Control. Real-time PCR was carried out using the QuantiFast Pathogen RT-PCR +IC Kit on the Rotor-Gene Q without any PCR optimization. Duplex reactions contained a fixed amount of Internal Control template. Each dilution was analyzed in triplicate; one replicate per dilution is shown.|Bovine herpes virus type 1 was serially diluted (100 to 10-4) and detected by either singleplex real-time PCR or by duplex real-time PCR in parallel with the Internal Control. Real-time PCR was carried out using the QuantiFast Pathogen PCR +IC Kit on the ABI 7500 according to the supplied protocols and without any PCR optimization. The duplex reactions contained a fixed amount of Internal Control template. Each dilution was analyzed in triplicate; one replicate is shown for each dilution.|[A] Q-Bond in QuantiFast Buffer allows the DNA polymerase and primer to bind as a single complex, reducing the annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing annealing time.|QuantiFast Pathogen +IC Kits provide a simple procedure for quantitative, multiplex, real-time PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, Mg2+, and Taq DNA polymerase, even for difficult assays (e.g., assays in which the copy number of the target gene is much smaller than that for the reference gene).|[A] NH4+ ions prevent nonspecific primers from annealing to the template. [B] Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template. Together with K+ and other cations, Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq Plus DNA Polymerase.|Simultaneous extraction and/or amplification of a pathogen target plus an internal positive control will rule out PCR inhibition or other problems that could give a false-negative result, leading to higher process safety. The QIAGEN Internal Control can be added prior to PCR amplification to provide an amplification control, or highly concentrated QIAGEN Internal Control can be added during nucleic acid extraction to provide both an extraction and an amplification control.|A 6-log range of both Norovirus RNA singleplex detection and Norovirus/IC duplex detection shows high precision and linearity. Error bars each represent ±1 SD of 3 real-time RT-PCR replicates.|Serial tenfold dilutions of RNA standards (in vitro transcribed RNA) were prepared using either QuantiTect Nucleic Acid Dilution Buffer or RNase-free water, as indicated. These dilutions were used as template in one-step RT-PCR either directly (0 test) or after storage for 2 or 4 weeks at -20°C. Using QuantiTect Nucleic Acid Dilution Buffer resulted in lower CT values and improved stability of the standards.|
Principle
To enable high process safety through the correct interpretation of negative results, each QuantiFast Pathogen _IC Kit contains reagents for multiplex, real-time detection of a user-defined pathogen target with the Internal Control. Amplifying control and target genes in the same reaction, instead of separate reactions, increases the reliability of gene quantification by minimizing handling errors.
QuantiFast Pathogen +IC Kits provide sensitive and rapid real-time multiplex PCR or one-step RT-PCR detection of pathogen nucleic acids 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. The specially developed fast PCR buffer contains the novel additive Q-Bond, which significantly reduces denaturation, annealing, and extension times (see figure "Fast primer annealing"). A balanced combination of K+ and NH4+ ions as well as unique synthetic Factor MP, 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.
| 5x QuantiFast Pathogen PCR Master Mix |
Concentrated master mix |
Highly concentrated and optimized for sensitive pathogen 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 |
| QuantiFast Pathogen 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 |
| Unique Q-Bond additive |
Faster PCR run times, enabling faster results and more reactions per day |
| Internal Control Assay |
Internal Control template |
Internal Control DNA template in the QuantiFast Pathogen PCR +IC Kit |
A universal DNA amplification control that can be used with different pathogen assays |
| Internal Control RNA in the QuantiFast Pathogen RT-PCR +IC Kit |
A universal RNA amplification control that can be used with different pathogen assays |
| Internal Control Assay |
Premixed primer/probe set (TaqMan® probe) labeled with MAX (equivalent to HEX, VIC, etc.) |
Will not interfere with primers against the pathogen-target |
| Additional kit components |
QuantiFast Pathogen RT Mix* |
Contains a unique formulation of Sensiscript Reverse Transcriptase |
Optimized for highly sensitive detection of pathogen RNA |
| ROX Dye Solution |
Separate tube of passive reference dye for normalization of fluorescent signals on Applied Biosystems 7500 real-time PCR systems. Optional: Can be used on Stratagene instruments from Agilent |
Precise quantification on cyclers that require ROX dye. Does not interfere with PCR on any real-time cycler |
| High-ROX Dye Solution |
Separate tube of passive reference dye for normalization of fluorescent signals on Applied Biosystems 7900 and StepOne real-time PCR systems |
| 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
QuantiFast Pathogen +IC Kits provide a simple procedure for the detection of a user-defined pathogen and the Internal Control. They contain a ready-to-use master mix for the real-time detection of viral RNA (1-step RT-PCR) or viral, bacterial, and fungal DNA (PCR). There is no need for optimization of reaction and cycling conditions. Simply mix the supplied master mix with the pathogen assay (primers and probe) and the supplied Internal Control Assay and the Internal Control DNA or RNA. Alternatively, if the Internal Control has been added to the sample purification procedure, add RNase-free water instead of Internal Control DNA or RNA to the reaction mix. Then add your sample DNA or RNA and start the reaction on any cycler. The kit handbook contains optimized protocols for use with TaqMan® probes on a wide range of cyclers. It also contains recommendations for selection of dye combinations.
Each QuantiFast Pathogen +IC Kit includes the Internal Control Assay and Internal Control DNA or RNA for use as an amplification control via direct addition to the reaction mix. Alternatively, the IC can be added to the purification procedure to control both the purification process and amplification. For addition of the Internal Control to the purification procedure, highly concentrated Internal Control DNA or RNA (High conc.) can be ordered separately (see figure "QIAGEN Internal Control").
Applications
QuantiFast Pathogen +IC Kits provide sensitive real-time PCR or one-step RT-PCR using sequence-specific probes for detection of pathogen DNA and/or RNA including an internal control. The kits can be used on a wide range of real-time cyclers, including cyclers from QIAGEN, Applied Biosystems, Bio-Rad, Roche (except for capillary cyclers), and Agilent.
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特点
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参数
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应用
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Pathogen Detection: Real-time PCR of viral, bacterial or fungal DNA (QuantiFast Pathogen PCR +IC Kit) or one-step RT-PCR for detection of viral RNA (QuantiFast Pathogen RT-PCR +IC Kit)
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反应类型
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Real-time PCR or one-step RT-PCR including of an internal control (IC)
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real-time或终点法PCR
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Real-time
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样本/目标类型
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QuantiFast Pathogen PCR +IC Kit: viral, bacterial or fungal DNA; QuantiFast Pathogen RT-PCR +IC Kit: viral RNA
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单一或多重
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Duplex
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SYBR Green I或序列特异性探针
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Sequence-specific probes
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热循环仪
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For most standard and fast real-time cylcers compatible with duplex PCR/RT-PCR, e.g. Rotor-Gene Q or cyclers from Agilent, Applied Biosystems, BioRad, Roche
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有/无ROX
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Master Mix is provided without ROX dye, but 2 separate ROX solutions are included: High-ROX Dye Solution for use with ABI cyclers except ABI 7500, ROX Dye Solution (low ROX conc.) for use with ABI 7500 and other suppliers
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图片
正确解读阴性结果。
在Rotor-Gene Q实时荧光定量PCR分析仪上采用内对照,在不同量的PCR抑制物(腐植酸) 存在的条件下,对两种浓度的病毒RNA靶点进行共扩增,重复两次。[A] 无模板对照 (NTC) 作为内对照信号对照。[B] 病毒RNA靶点和内对照的扩增证明了扩增的成功。[C] 内对照显示有少量的抑制物存在。[D] 内对照检测失败说明在抑制物存在时扩增反应失败。
在Rotor-Gene Q实时荧光定量PCR分析仪上灵敏检测BHV-1。
利用单重real-time PCR或双重real-time PCR检测梯度稀释的牛疱疹病毒1型 (100至10-4) 及内对照。在Rotor-Gene Q实时荧光定量PCR分析仪上使用QuantiFast Pathogen PCR +IC Kit,参照试剂盒提供的实验方案进行real-time PCR,无需PCR优化。双重反应中包含固定量的内对照模板。重复分析各稀释度三次;如图显示了每种稀释度样本各一个。
灵敏检测Norovirus。
利用单重real-time RT-PCR或双重real-time PCR检测梯度稀释的诺如病毒RNA转录本 (100至10-5) 及内对照。在Rotor-Gene Q实时荧光定量PCR分析仪上使用QuantiFast Pathogen RT-PCR +IC Kit进行real-time PCR,无需PCR优化。双重反应中包含固定量的内对照模板。重复分析各稀释度三次;如图显示了每种稀释度样本各一个。
在ABI 7500上灵敏检测BHV-1。
利用单重real-time PCR或双重real-time PCR检测梯度稀释的牛疱疹病毒1型 (100至10-4) 及内对照。在ABI 7500上使用QuantiFast Pathogen PCR +IC Kit,参照试剂盒提供的实验方案进行real-time PCR,无需PCR优化。双重反应中包含固定量的内对照模板。重复分析各稀释度三次;如图显示了每种稀释度样本各一个。
退火时引物快速结合。
[A] QuantiFast Buffer中的Q-Bond能使DNA聚合酶和引物结合为单一复合物,将退火时间缩短至几秒。此外,独特的缓冲液组分可支持DNA的熔解,缩短变性和延伸时间。[B] 若无Q-Bond,引物和聚合酶会相继与模板结合,退火时间较长。
QIAGEN多重试剂盒。
QuantiFast Pathogen +IC Kit提供了简单的多重定量real-time PCR步骤。与当前的方法相比,即便是进行复杂的分析(如目的基因的拷贝数明显少于对照基因的分析),该试剂盒亦无需优化引物、Mg2+和Taq DNA聚合酶的浓度,甚至是困难模板(例如:靶基因拷贝数大大少于参考基因)。
独特的PCR缓冲液。
[A] NH4+防止引物与模板的非特异性结合。[B] MP合成因子是创新的PCR添加剂,能够提高模板处局部的引物浓度,与K+和其他离子一起,MP合成因子能够特异性稳定结合的引物,使HotStarTaq Plus DNA Polymerase作用下的引物延伸更加高效。
QIAGEN Internal Control。
同时抽提和/或扩增病原体靶点和内部阳性对照可以消除PCR抑制或其他可导致假阳性结果的问题,提高过程的安全性。在PCR扩增前加入QIAGEN Internal Control可提供扩增对照,或者在核酸抽提过程中加入高浓度的QIAGEN Internal Control,提供抽提和扩增对照。
单重和多重检测都具有高度线性契合度和准确性。
诺如病毒RNA单重检测和诺如病毒/IC双重检测的6-log范围显示了高精度和线性度。3次real-time RT-PCR重复结果误差为±1 SD。
对RNA标准品进行可靠的稀释和储存。
使用QuantiTect Nucleic Acid Dilution Buffer或不含RNA酶的纯水按说明制备连续10倍梯度稀释的RNA标准品(体外转录的RNA)。直接使用这些稀释样本作为模板或将其置于-20°C保存2或4周后进行一步法RT-PCR。使用QuantiTect Nucleic Acid Dilution Buffer稀释标准品可获得更低的CT值和更佳的稳定性。
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