QuantiFast Multiplex PCR Kits
For fast, multiplex, real-time PCR and two-step qRT-PCR using sequence-specific probes
- Sensitive detection of up to 4 targets in 1 tube
- Faster results with time savings of up to 50%
- Successful multiplex PCR without the need for optimization
- Reliable quantification of low- and high-abundance targets
QuantiFast Multiplex PCR Kits enable fast and reliable quantification of up to 4 cDNA or gDNA targets in a single tube by multiplex, real-time PCR or two-step RT-PCR. Q-bond technology and an optimized master mix promote fast multiplex real-time PCR, not only on fast cyclers with short ramping times, but also on standard cyclers. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qPCR on any real-time cycler without the need for optimization. Two kit formats are available: the QuantiFast Multiplex PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiFast Multiplex PCR +R Kit for all other cyclers. For convenience, the master mix can be stored at 2–8°C.
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QuantiFast Multiplex PCR Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast Multiplex PCR Master Mix (with ROX dye), 2 x 2 ml RNase-Free Water
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204654
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QuantiFast Multiplex PCR Kit (2000)
For 2000 x 25 µl reactions: 25 ml 2x QuantiFast Multiplex PCR Master Mix (with ROX dye), 20 ml RNase-Free Water
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204656
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QuantiFast Multiplex PCR +R Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast Multiplex PCR Master Mix (without ROX dye), 210 µl ROX Dye Solution, 2 x 2 ml RNase-Free Water
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204754
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QuantiFast Multiplex PCR +R Kit (2000)
For 2000 x 25 µl reactions: 25 ml 2x QuantiFast Multiplex PCR Master Mix (without ROX dye), 1.05 ml ROX Dye Solution, 20 ml RNase-Free Water
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204756
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QuantiFast Multiplex PCR +R Kit (4000)
For 4000 x 25 µl reactions: 2 x 25 ml 2x QuantiFast Multiplex PCR Master Mix (without ROX dye), 2 x 1.05 ml ROX Dye Solution, 20 ml RNase-Free Water
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204757
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QuantiFast Multiplex PCR 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。
PCR时间极大缩短。|灵敏的PCR,动态范围宽泛。|三重PCR和单重PCR的结果相当。|高度灵敏性。|退火时引物快速结合。|QIAGEN多重试剂盒。|独特的PCR缓冲液。|模板量降低,CT值仍呈线性。|
使用QuantiFast Multiplex Kit进行两步法real-time RT-PCR,可缩短PCR总运行时间达50%(40个循环运行;与QuantiTect Multiplex Kit相比较)。I:iCycler iQ;L1:LightCycler 480;L2:LightCycler 2.0;A1:ABI PRISM 7900;A2:Applied Biosystems 7500 Fast System;M:Mx3005P。|使用DNA模板混合物在Applied Biosystems 7500 Fast System上进行两次重复反应,混合物中包含108个拷贝的β-actin(数据在插图中显示)和106至10个拷贝的RPS27A (核糖体蛋白)。[A] QuantiFast Multiplex PCR +R Kit的灵敏度明显高于[B] Supplier AII的双重PCR试剂盒,使得在快速循环模式下运行的循环仪能够检测低至10拷贝的靶点,并定量超过6 log稀释范围的模板。|在LightCycler 480上使用QuantiFast Multiplex PCR +R Kit和采用Primer Express设计的针对 [A] RPS27A (一种核糖体蛋白)、[B] GAPDH (管家基因) 和 [C] UBC (管家基因) 的TaqMan Assay进行三重和单重PCR。模板为HeLa细胞cDNA (10 ng、1 ng或0.1 ng),进行两次重复反应。三重PCR (彩色曲线) 和单重PCR (灰色曲线) 的比较CT值证明了在分析不同丰度的靶点时,使用QuantiFast Multiplex PCR +R Kit进行三重PCR的可靠性。|使用QuantiFast Multiplex PCR +R Kit和采用Primer Express设计的TaqMan Assay在Applied Biosystems 7500 Fast System上进行4重real-time PCR。使用10 ng Ramos细胞系cDNA进行三次重复反应。|[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。|QuantiFast Multiplex PCR 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。|[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。|在Applied Biosystems 7500 Fast System上使用QuantiFast Multiplex PCR +R Kit及针对t (8;14) 染色体易位和GAPDH的分析进行双重和单重PCR。使用Ramos细胞系的基因组DNA为模板 (10 ng至0.625 ng的两倍稀释),进行四重反应。在 [A] 单重和 [B] 双重反应中,CT值随着模板稀释度的降低呈线性升高,上升幅度为1个CT值,说明该试剂盒能够精确区分模板量的微小差异。|
性能
The special master mix supplied with QuantiFast Multiplex PCR Kits allows rapid setup of multiplex reactions and delivers successful results at the first attempt, providing multiplex PCR data that are comparable with singleplex PCR data (see figure "Comparable results in triplex and singleplex PCR"). The kits can clearly distinguish between small differences in the amount of template. Even with twofold differences in template amount, QuantiFast Multiplex PCR Kits provide accurate quantification of targets of widely differing abundance (see figure "Linear CT values over twofold decreases in template").
QuantiFast Multiplex PCR Kits reduce PCR run times by up to 50%, allowing you to get results significantly faster (see figure "Significantly reduced PCR times"). As little as 10 copies of target can be detected in just 60 minutes (see figure "Sensitive duplex PCR and a wide dynamic range"). You can also greatly increase your sample throughput or efficiently share a cycler with other users. Fast results in multiplex, real-time PCR of up to 4 targets are achieved without compromising performance (see figure "Uncompromised sensitivity").
原理
QuantiFast Multiplex PCR Kits deliver highly sensitive and rapid results over a wide dynamic range on both standard and fast cyclers without optimization (see flowchart "QIAGEN multiplex kits"). 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").
Amplifying reference and target genes in the same reaction instead of in separate reactions increases the reliability of gene quantification by minimizing handling errors. The QuantiFast Multiplex PCR Buffer includes a balanced combination of K+ and NH4+ ions as well as the unique synthetic Factor MP, which together 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, HotStarTaq Plus DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.
| HotStarTaq Plus DNA Polymerase |
5 min activation at 95ºC |
Set up of qPCR reactions at room temperature |
| QuantiFast Multiplex PCR 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 |
| ROX dye† |
Normalizes fluorescent signals on Applied Biosystems and, optionally, Agilent instruments |
Precise quantification on cyclers that require ROX dye. Does not interfere with PCR on any real-time cycler |
操作流程
QuantiFast Multiplex PCR Kits contain ready-to-use master mixes that eliminate the need for optimization of reaction and cycling conditions. Simply add template DNA and primer-probe sets to the master mix and follow the protocol in the handbook to get fast and reliable results on any real-time cycler. Kits are available with or without ROX passive reference dye in the master mix, enabling use on virtually any real-time cycler (see table). Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.
| Supplied in master mix |
QuantiFast Multiplex PCR Kit |
All cyclers from Applied Biosystems except Applied Biosystems 7500 |
| Supplied in separate tube |
QuantiFast Multiplex PCR +R Kit |
Applied Biosystems 7500 and cyclers from Bio-Rad, Cepheid, Eppendorf, QIAGEN, Roche, Agilent, and other suppliers |
For optimal results in real-time two-step RT-PCR, we recommend synthesizing cDNA using the QuantiTect Reverse Transcription Kit, which provides fast cDNA synthesis in just 20 minutes with integrated removal of genomic DNA contamination.
QuantiFast Probe Assays are predesigned, genomewide assays that use hydrolysis, probe-based detection. They are delivered with the QuantiFast Multiplex PCR Kit for guaranteed results in duplex, two-step real-time RT-PCR.
应用
QuantiFast Multiplex PCR Kits can be used for multiplex gene expression analysis of cDNA or gDNA targets on any real-time cycler. This includes instruments from Agilent, Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, and Roche. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene Multiplex PCR Kit, which has been specially developed for fast cycling on these instruments.
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特点
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参数
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应用
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Quantification of cDNA or genomic DNA targets in a multiplex real-time PCR
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反应类型
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Real-time two-step RT-PCR
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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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cDNA, DNA
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单一或多重
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Multiplex
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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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Real-time cyclers dedicated for multiplex PCR (e.g., most Applied Biosystems real-time PCR cyclers, Roche LightCycler® 480, iCycler iQ®, Rotor-Gene)
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有/无ROX
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Available with ROX in master mix and with ROX as separate vial
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FAQ ID -1430
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FAQ ID -589
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FAQ ID-751
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FAQ ID -1979
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For quantitative, multiplex, real-time PCR and two-step RT-PCR with fast cycling using sequence-specific probes
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显示更多
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图片
PCR时间极大缩短。
使用QuantiFast Multiplex Kit进行两步法real-time RT-PCR,可缩短PCR总运行时间达50%(40个循环运行;与QuantiTect Multiplex Kit相比较)。I:iCycler iQ;L1:LightCycler 480;L2:LightCycler 2.0;A1:ABI PRISM 7900;A2:Applied Biosystems 7500 Fast System;M:Mx3005P。
灵敏的PCR,动态范围宽泛。
使用DNA模板混合物在Applied Biosystems 7500 Fast System上进行两次重复反应,混合物中包含108个拷贝的β-actin(数据在插图中显示)和106至10个拷贝的RPS27A (核糖体蛋白)。[A] QuantiFast Multiplex PCR +R Kit的灵敏度明显高于[B] Supplier AII的双重PCR试剂盒,使得在快速循环模式下运行的循环仪能够检测低至10拷贝的靶点,并定量超过6 log稀释范围的模板。
三重PCR和单重PCR的结果相当。
在LightCycler 480上使用QuantiFast Multiplex PCR +R Kit和采用Primer Express设计的针对 [A] RPS27A (一种核糖体蛋白)、[B] GAPDH (管家基因) 和 [C] UBC (管家基因) 的TaqMan Assay进行三重和单重PCR。模板为HeLa细胞cDNA (10 ng、1 ng或0.1 ng),进行两次重复反应。三重PCR (彩色曲线) 和单重PCR (灰色曲线) 的比较CT值证明了在分析不同丰度的靶点时,使用QuantiFast Multiplex PCR +R Kit进行三重PCR的可靠性。
高度灵敏性。
使用QuantiFast Multiplex PCR +R Kit和采用Primer Express设计的TaqMan Assay在Applied Biosystems 7500 Fast System上进行4重real-time PCR。使用10 ng Ramos细胞系cDNA进行三次重复反应。
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 Multiplex PCR 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。
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。
模板量降低,CT值仍呈线性。
在Applied Biosystems 7500 Fast System上使用QuantiFast Multiplex PCR +R Kit及针对t (8;14) 染色体易位和GAPDH的分析进行双重和单重PCR。使用Ramos细胞系的基因组DNA为模板 (10 ng至0.625 ng的两倍稀释),进行四重反应。在 [A] 单重和 [B] 双重反应中,CT值随着模板稀释度的降低呈线性升高,上升幅度为1个CT值,说明该试剂盒能够精确区分模板量的微小差异。
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