Certal Residual DNA Detection Kits

基于探针技术灵敏检测残留的宿主细胞DNA,包含内参

  • 卓越的重复性,最小限度定量
  • 线性范围宽
  • 内参可靠准确
  • 优化的方案,适用于多种real-time PCR仪
  • 完整的试剂盒,包括内参和阳性对照

在生物制药中,评估最终的治疗材料确保无污染物质至关重要,如来自宿主细胞的残留DNA。Certal Residual DNA Detection Kits使用双标记水解探针(即TaqMan探针),通过real-time PCR灵敏和快速的定量残留的宿主细胞基因组DNA。Certal CHO Detection Kit包含进行双重real-time PCR的试剂,用于检测CHO残留的宿主细胞DNA,以及Certal Internal Control。该试剂盒还包含Certal Residual DNA Positive Control,用于存在CHO宿主细胞DNA信号时验证PCR分析的功能,以及2种不同浓度的ROX阴性对照染料,能够用于几乎所有的real-time仪器。

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Certal CHO Detection Kit (100)
For 100 x 25 µl reactions: 1.25 ml Certal Residual DNA PCR Master Mix, Certal Internal Control DNA and Assay, CHO DNA Positive control and CHO-Assay, 100 µl Uracil-N-Glycosylase, 210 µl High-ROX Dye Solution, 210 µl ROX Dye Solution, Nucleic Acid Dilution Buffer, TE Buffer
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Reliable detection of the QIAGEN Internal Control.|Certal Residual DNA Detection Kit procedure.|Sensitive and reliable detection of CHO genomic DNA on the Rotor-Gene Q.|Low limit of detection.|Sensitive and reliable detection on a wide range of cyclers.|Highly sensitive and reliable detection.|
Primers and probe for the target (CHO) assay and the internal control assay are added to the Certal ResDNA Master Mix. Once internal control template and sample template is added and reactions are distributed, they can be run using a wide range of cyclers. Optionally, positive CHO controls can be tested, and UNG may be used to control for carryover contamination of PCR products.|Primers and probe for the target (CHO) assay and the internal control assay are added to the Certal ResDNA Master Mix. Once internal control template and sample template is added and reactions are distributed, they can be run using a wide range of cyclers. Optionally, positive CHO controls can be tested, and UNG may be used to control for carryover contamination of PCR products.|The reliability of the Certal Residual DNA Assay on the Rotor-Gene Q is demonstrated by evenly spaced amplification plots for the target signal and stable CT values for the internal control (inset), which indicate that sensitive detection of the internal control is not affected by the wide range of CHO genomic DNA concentration.|The limit of detection (LOD), defined as the lowest concentration with 100% positive amplification result for 24 replicates, was determined to be 1 fg using the Certal CHO Detection Kit Assay. The limit of quantification (LOQ), defined as the lowest concentration of a calculated unknown within ±0.5 CT of the standard value, is 10 fg.|Target DNA ranging from 3 fg to 30 ng was analyzed using the Certal CHO Detection Kit on a wide range of commonly used cyclers. The high linear range of detection demonstrates the reliability and adaptability of Certal assays on several different qPCR cyclers.|CHO DNA (0.3 fg to 300 ng) was analyzed using the Certal CHO Detection Kit on the Applied Biosystems ABI 7900 HT (384-well format). Highly sensitive detection was achieved with a low limit of quantification and a high linear range of detection, ranging from 10 fg to 300 ng. Error bars, demonstrating the reliability of the reaction, each represent ±1 SD of 4 replicates and are smaller than data points where not visible. LOQ: Limit of quantification, defined as the lowest concentration of a calculated unknown within ±0.5 CT of the standard value. This was determined experimentally: the mean of the unknowns were not permitted to deviate more than 0.5 CT from the mean of the standard curve. LOD: Limit of detection, defined as the lowest concentration with 100% positive amplification result for 24 replicates.|
Performance

Certal Residual DNA Detection Kits能够灵敏、可靠的定量检测残留的基因组DNA(gDNA),这对生产安全和可靠的生物制品至关重要(参见"Highly sensitive and reliable detection")。检测残留的DNA需要最小限度的检测和高线性范围,以确保可靠的检测,即使残留的gDNA的水平很低(参见"Low limit of detection")。Certal Residual DNA Detection Kits与多种循环仪兼容,提供灵敏、可靠的数据(参见"Sensitive and reliable detection on a wide range of cyclers"和"Sensitive and reliable detection of CHO genomic DNA on the Rotor-Gene Q")。

为确保安全,Certal Residual DNA Detection Kits包含QIAGEN Internal Control,专门设计用于正确解读阴性结果且不干预目标分析。QIAGEN Internal Control与基因组DNA共扩增时能够可靠的检测到(参见"Reliable detection of the QIAGEN Internal Control")。

Principle

为了确保最终的治疗材料中无来自宿主细胞的污染物,该宿主细胞用于生产生物制品,Certal Residual DNA Detection Kits运行预订的分析特异性检测CHO宿主细胞DNA。Certal Residual DNA Assay由优化的引物对和一个用于可靠检测残留的CHO基因组DNA的FAM标记的水解探针组成。人类DNA和Certal Internal Control DNA不会干扰Certal Residual DNA Assay。

预混的Certal Internal Control Assay包括正向反向引物和用于检测Certal Internal Control DNA的MAX标记的水解探针。Certal Internal Control Assay已经过特殊优化,可防止Certal Residual DNA Assays的干扰。Certal Residual DNA Positive Control通过检测扩增反应本身的功能,能够正确解读阴性结果。

Certal ResDNA PCR Buffer已经过优化,用于灵敏、可靠的定量双重real-time PCR结果。预混液还包括dUTP,能够在开始PCR前用尿嘧啶正糖苷酶预处理,以确保污染的PCR产物不影响随后的PCR反应。

Certal Residual DNA Detection Kit的组成
组分特征 优势
Certal Residual DNA Assays和DNA对照
50x Certal Residual DNA Assay 预订的、经优化和验证的引物-探针组(FAM标记) 可靠检测残留的CHO宿主细胞DNA
Certal Residual DNA阳性对照 由Certal Residual DNA Assay扩增的阳性对照DNA 在检测不到残留的宿主细胞DNA时,验证Certal Residual DNA Assay是正确工作的
50x Certal Internal Control Assay 预订的、经优化和验证的引物-探针组(MAX标记)用于Certal Internal Control DNA检测 只特异性检测Certal Internal Control DNA。经优化防止Certal Residual DNA Assays的干扰
Certal Internal Control DNA 由Certal Internal Control Assay扩增的Internal Control DNA 排除PCR抑制剂存在或在纯化过程中控制纯化和扩增
Certal ResDNA PCR Master Mix
HotStarTaq DNA Polymerase    95°C条件下激活15 分钟 室温下设置qPCR反应
Certal ResDNA PCR Buffer KCl和(NH4)2SO4的平衡组合 特异性引物退火,确保可靠的PCR结果
Synthetic Factor MP 同一管中可靠的多重分析4个基因
dNTP mix 包括dUTP,部分取代dTTP和可选的UNG 处理 当反应使用尿嘧啶正糖苷酶(UNG)预处理时,有效去除PCR产物的遗留
外加组分
50x ROX Dye Solution 含阴性对照染料的分离管,用于标准化在Applied Biosystems 7500 real-time PCR体系上的荧光信号。可选:可用于来自Agilent的Stratagene仪器 准确定量需ROX染料的循环。不干扰在任何real-time循环仪上的PCR
50x High-ROX Dye Solution 含阴性对照染料的分离管,用于标准化在Applied Biosystems 7900和StepOne real-time PCR体系上的荧光信号
Uracil-N-Glycosylase (UNG) 纯化的、无核酶的重组酶,在分离管中 有效去除PCR产物的遗留
QuantiTect Nucleic Acid Dilution Buffer 专有的缓冲配方,用于稀释和贮存核酸标准品 在稀释和反应建立过程中稳定RNA和DNA标准品,以及防止塑料表面核苷酸的损失,如管子和枪头
Certal Residual DNA Detection Kit还包括用于重组冻干制剂分析的Buffer TE和RNase-free水。
Procedure

Certal Residual DNA Detection Kits提供简单的流程用于检测宿主细胞DNA,包含Certal Internal Control和Certal Residual DNA Positive Control(参见"Certal Residual DNA Detection Kit procedure")。只需简单的将预混液与Certal Residual DNA Assay、Certal Internal Control Assay和Certal Internal Control DNA混合,然后加入DNA样本或Certal Residual DNA Positive Control。该试剂盒的操作手册上有优化的方案,用于多种循环仪。ROX提供不同浓度的2管,用于多种real-time仪器(参见下表)。

选择正确的ROX浓度
ROX染料溶液
仪器 不需要 ROX染料溶液 高浓度ROX染料溶液
Rotor-Gene Q实时荧光定量PCR分析仪 X
Rotor-Gene 6000和Rotor-Gene 3000 X
Applied Biosystems 7500和ABI ViiA7 X
Applied Biosystems 7900HT、StepOne and StepOne Plus、ABI PRISM 7700、7000和7300 X
Mx3000P、Mx3005P和Mx4000 X 可选
iCycler iQ System X
LightCycler 480 X
Applications

Certal Residual DNA Detection Kits使用用于检测宿主细胞DNA的序列特异性探针进行灵敏的real-time PCR,包括内参。该试剂盒可用于多种real-time PCR仪,包括来自QIAGEN、Applied Biosystems、Bio-Rad、Roche和Agilent的PCR仪。

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Kit Handbooks
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For sensitive real-time PCR detection of residual host cell DNA and an internal control using sequence-specific probes
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