灵敏、可靠的检测病毒RNA/DNA和细菌DNA,含有内参

  • 同时检测目标病原体和内参
  • 5x预混液具有更高的灵敏度,且起始样本量更大
  • 正确解读阴性信号,检测灵敏度更高
  • 清晰检测微弱的阳性信号
  • 快速的通用型实验方案适用于标准和快速PCR仪

QuantiFast Pathogen +IC Kits使用序列特异性探针,可灵敏、快速的对病原体核酸进行real-time PCR或一步法RT-PCR检测。该试剂盒含有检测4个用户定义的病原体核酸(如:病毒、细菌、真菌等)所需的试剂和内参,可对阴性结果进行正确解读,提高了检测灵敏度。该试剂盒有两种规格。QuantiFast Pathogen RT-PCR +IC Kit含有RNA内参模板和内参引物/探针对,用于检测病毒RNA。QuantiFast Pathogen PCR +IC Kit含有DNA内参模板和内参引物/探针对,用于检测病毒、细菌或真菌DNA。这两种试剂盒均提供分装在两个管中的不同浓度的ROX染料,因此适用于多种real-time PCR仪。为便于使用,预混液可存放在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
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
211354
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QuantiFast Pathogen RT-PCR +IC Kit (100)
For 100 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
211452
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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
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
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
211492
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QuantiFast Pathogen +IC Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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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.|
Performance

QuantiFast Pathogen +IC Kits可通过多重扩增,同时检测病毒RNA或DNA、以及自带的内参(参见"Sensitive detection of Norovirus on the Rotor-Gene Q"和"High linearity and precision of singleplex and duplex detection")。实验方案专为快速PCR设计,适用于多数PCR仪,具有极高的可靠性(参见"Sensitive detection of BHV-1 on the Rotor-Gene Q"和"Sensitive detection of BHV-1 on the ABI 7500")。使用QuantiFast Pathogen +IC Kits同时扩增目标病原体和内参,提高了检测的可靠性,能够确保正确解读阴性结果(参见"Correct interpretation of negative results")。 

该试剂盒提供QuantiTect Nucleic Acid Dilution Buffer,可在稀释和反应体系构建时稳定RNA和DNA标准品,避免核酸损失。能够可靠的稀释标准品,进而用于核酸的定量检测,检测范围广,覆盖从低至高的CT值。该缓冲液能够确保标准品长期保存不降解(参见"Reliable dilution and storage of RNA standards")。

Principle

为避免假阴性结果,所有QuantiFast Pathogen +IC Kit都含有必须的试剂,可real-time检测病原体和内参。在同一反应中扩增内参和靶基因,提高了定量检测的可靠性,减少了手动操作可能产生的错误。

QuantiFast Pathogen +IC Kits可通过多重PCR灵敏、快速的进行real-time检测,或通过一步法RT-PCR检测病原体(参见"QIAGEN multiplex kits")。优化的预混液可确保PCR产物在多重扩增反应中以相同的效率和灵敏度扩增。特别研制的快速PCR缓冲液含有新型的添加剂Q-Bond,能够大幅度缩短变性、退火和延伸的时间(参见"Fast primer annealing")。浓度平衡的K+和NH4+离子、以及独特的Factor MP能够促进引物和探针与核酸模板特异性结合,确保PCR效率高(参见"Unique PCR buffer")。此外,配方独特的Sensiscript Reverse Transcriptase能确保病毒RNA的高度灵敏的逆转录,HotStarTaq Plus DNA Polymerase则提供严格的热启动,防止非特异性产物的形成。

QuantiFast Pathogen +IC Kit的组分
试剂盒组分特点 优势
5x QuantiFast Pathogen PCR Master Mix 浓缩的预混液 浓度高,专为灵敏的检测病原体而设计 可加入的模板体积更多,增加了灵敏度
HotStarTaq Plus DNA Polymerase 95ºC孵育5分钟激活 在室温进行qPCR反应体系构建
QuantiFast Pathogen Buffer 浓度平衡的NH4+和K+离子 引物探针的特异性结合确保获得可靠结果
合成的Factor MP 在单管中对至多4个基因进行多重分析
独特的Q-Bond添加物 PCR运行时间缩短,更快获得结果,一天内可完成更多PCR反应
Internal Control Assay Internal Control 模板 QuantiFast Pathogen PCR +IC Kit含有内参DNA模板 通用的DNA扩增对照能用于分析不同的病原体
QuantiFast Pathogen RT-PCR +IC Kit含有内参RNA 通用的RNA扩增对照能用于分析不同的病原体
Internal Control Assay 引物探针预混液(TaqMan探针)标记有MAX(相当于HEX、VIC等) 不会干扰引物
Additional kit components QuantiFast Pathogen RT Mix* 含有配方独特的Sensiscript Reverse Transcriptase 专为灵敏检测病原体RNA而设计
ROX Dye Solution 参照染料可用于Applied Biosystems 7500 real-time PCR仪的荧光信号校准。可选:可用于Stratagene仪器 准确定量检测需要使用ROX染料。不会干扰PCR反应
High-ROX Dye Solution 参照染料可用于Applied Biosystems 7900 PCR仪和StepOne real-time PCR仪的荧光信号校准
QuantiTect Nucleic Acid Dilution Buffer 缓冲液为专有配方,用于稀释和储存核酸标准品 在稀释和反应体系构建时稳定RNA和DNA标准品,避免核酸损失
* 仅QuantiFast Pathogen RT-PCR +IC Kits提供
Procedure

QuantiFast Pathogen +IC Kits通过简单的流程,即可检测病原体和内参。试剂盒含有即用型预混液,可real-time检测病毒RNA(一步法RT-PCR)或病毒、细菌和真菌DNA(PCR)。无需优化反应条件。只需将预混液与检测试剂(引物与探针)、Internal Control Assay和Internal Control DNA或RNA 混合。也可在样本纯化步骤中加入内参,然后加入不含RNase的纯水至反应混合液。将样本DNA或RNA加入后即可开始反应,适用于各种PCR仪。试剂盒使用手册含有优化的实验方案,适用于TaqMan探针和多种PCR仪。使用手册中还推荐了可用的染料。

所有QuantiFast Pathogen +IC Kit都含有Internal Control Assay和Internal Control DNA或RNA,可直接加入反应混合液,用作扩增对照。此外,也可在纯化过程中加入内参,作为纯化和扩增的对照。如需在纯化过程中加入内参,可另外订购高浓度的Internal Control DNA或RNA (High conc.) (参见"QIAGEN Internal Control")。

Applications

QuantiFast Pathogen +IC Kits利用序列特异性探针进行灵敏的real-time PCR或一步法RT-PCR,检测病原体DNA和/或RNA,以及内参。该试剂盒适用于多种real-time PCR仪,包括QIAGEN、Applied Biosystems、Bio-Rad、Roche和Agilent的仪器。

Feature
Specifications
Applications 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)
Reaction type Real-time PCR or one-step RT-PCR including of an internal control (IC)
Real-time or endpoint Real-time
Sample/target type QuantiFast Pathogen PCR +IC Kit: viral, bacterial or fungal DNA; QuantiFast Pathogen RT-PCR +IC Kit: viral RNA
Single or multiplex Duplex
SYBR Green I or sequence-specific probes Sequence-specific probes
Thermal cycler 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
With or without ROX 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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