QuantiFast Pathogen +IC Kits
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
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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 (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
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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
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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 are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
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
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。
| 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 |
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。
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Download Safety Data Sheets for QIAGEN product components.
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Download Safety Data Sheets for QIAGEN product components.
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Images
Correct interpretation of negative results.
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.
Sensitive detection of BHV-1 on the Rotor-Gene Q.
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.
Sensitive detection of Norovirus.
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.
Sensitive detection of BHV-1 on the ABI 7500.
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.
Fast primer annealing.
[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.
QIAGEN multiplex kits.
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).
Unique PCR buffer.
[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.
QIAGEN Internal Control.
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.
High linearity and precision of singleplex and duplex detection.
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.
Reliable dilution and storage of RNA standards.
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.
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