Certal Residual DNA Detection Kits

For sensitive probe-based detection of residual CHO host cell DNA, including an internal control

  • Low limit of quantification with excellent reproducibility
  • High linear range
  • Reliable and precise internal control
  • Optimized protocols for a broad range of real-time cyclers
  • Complete kits including internal control and positive control

When working with biopharmaceuticals, it is essential to evaluate the final therapeutic material to ensure that it is free of contaminating substances, such as residual DNA from the host cell. Certal Residual DNA Detection Kits are designed for sensitive and rapid real-time PCR quantification of residual host cell genomic DNA using dual-labeled hydrolysis (also known as TaqMan®) probes. The Certal CHO Detection Kit contains reagents for duplex real-time PCR detection of CHO residual host cell DNA, plus the Certal Internal Control. The kit also includes the Certal Residual DNA Positive Control, which verifies the functionality of the PCR assay in the absence of a signal from CHO host cell DNA, and 2 formats of ROX passive reference dye in different concentrations, enabling use on virtually any real-time instrument.

製品名 Cat. no. List price:
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
211822
¥213,000

The Certal Residual DNA Detection Kits are intended for molecular biology applications in pharmaceutical product testing. These products are not intended for the diagnosis, prevention, or treatment of a disease.

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Certal Residual DNA Detection Kit操作手順|Certal Residual DNA Detection Kit procedure.|Rotor-Gene QでCHOゲノムDNAの感度と信頼性の高い検出|低い検出限界|様々なサイクラーで感度と信頼性の高い検出|感度および信頼性の高い検出|
ターゲット(CHO)アッセイ用のプライマーとプローブとインターナルコントロールをCertal ResDNA Master Mixに添加した。インターナルコントロールおよびサンプルのテンプレートを添加し、反応溶液を分注すれば、様々なサイクラーで反応を開始できる。オプションでポジティブCHOコントロールがテストと、PCR産物の混入をコントロールするためにUNGを使用もできる。|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.|Rotor-Gene QでのCertal Residual DNA Assayの信頼性の高いことが、ターゲットシグナルの増幅プロットが等間隔であること、およびインターナルコントロールの安定したCT値により証明され(挿入図)、インターナルコントロールの高感度検出がCHOゲノムDNAに影響されないことを示す。|24 replicatesで100% ポジティブな増幅結果が得られる最少濃度として定義された検出限界(LOD)は、Certal CHO Detection Kit Assayを使用して1 fgであった。標準値の±0.5 CT 内にある未知数の計算値の最少濃度として定義された定量限界(LOQ)は10 fgである。|3 fgから30 ngのターゲットDNAを、Certal CHO Detection Kitを用いて汎用されている様々なサイクラーで解析した。広範囲にわたる検出の直線性は、いくつかの異なるqPCRサイクラーでCertal アッセイが信頼性と適応性のあることを証明した。|[A] Vero DNA(0.3 fg ~30 ng) あるいは [B] CHO DNA(0.3 fg~300 ng) を、相当するCertal Residual DNA Detection Kitを用いてApplied Biosystems ABI 7900 HT(384ウェルフォーマット)で解析した。高感度検出が低い定量限界と広い直線性の検出範囲で可能であった:[A] 3 fgから30 ngおよび [B] 3 fgから300 ng。反応の信頼性を示すエラーバーは4 replicates実験の±1 SDを示しているが、データポイントよりも小さいエラーバーは表示されていない。 LOQ:標準値の±0.5 CT内にある未知数の計算値の最少濃度として定義された定量限界。これは実験的に決められた:未知数の平均値は標準曲線の平均よりも0.5 CT 以上ずれていなかった。 LOD:検出限界、24 replicatesで100% ポジティブな増幅結果が得られる最少濃度として定義される。|
パフォーマンス

Certal Residual DNA Detection Kits enable sensitive and reliable detection and quantification of residual genomic DNA (gDNA), which is essential for the production of safe and reliable biopharmaceuticals (see figure "Highly sensitive and reliable detection"). The Certal CHO Detection Kit detects residual gDNA from Chinese Hamster Ovary (CHO) cells with a low limit of detection and high linear range, ensuring reliable detection of even low levels of residual gDNA (see figure "Low limit of detection"). Certal Residual DNA Detection Kits are compatible with a wide range of cyclers, providing sensitive and reliable data (see figures "Sensitive and reliable detection on a wide range of cyclers" and "Sensitive and reliable detection of CHO genomic DNA on the Rotor-Gene Q").

For high process safety, Certal Residual DNA Detection Kits include the QIAGEN Internal Control, which is specifically designed to enable the correct interpretation of negative results and to not interfere with the target assay. The QIAGEN Internal Control is reliably detected when coamplified with genomic DNA (see figure "Reliable detection of the QIAGEN Internal Control").

原理

To ensure that contaminating substances from the host cell line used to produce biopharmaceuticals are absent from final therapeutic material, Certal Residual DNA Detection Kits deliver a predesigned assay to specifically detect CHO host cell DNA. Each Certal Residual DNA Assay consists of an optimized primer pair and a FAM labeled hydrolysis probe for reliable detection of residual CHO genomic DNA. Human background DNA and Certal Internal Control DNA will not interfere with the Certal Residual DNA Assay.

The premixed Certal Internal Control Assay contains a forward and reverse primer and a MAX labeled hydrolysis probe for the detection of Certal Internal Control DNA. It has been specifically optimized to prevent interference with Certal Residual DNA Assays. The Certal Residual DNA Positive Control enables the correct interpretation of negative results by testing the functionality of the amplification reaction itself.

Certal ResDNA PCR Buffer has been optimized to deliver sensitive and reliable quantitative, duplex real-time PCR results. The master mix also contains dUTP, enabling pretreatment with uracil-N-glycosylase (UNG) prior to starting PCR to ensure that any contaminating PCR products do not affect subsequent PCR reactions.

Certal Residual DNA Detection Kit Components
ComponentFeatures Benefits
Certal Residual DNA Assays and control DNA
50x Certal Residual DNA Assay Predesigned, optimized, and validated primer-probe set (FAM labeled) Reliable detection of residual CHO host-cell DNA
Certal Residual DNA Positive Control Positive control DNA that will be amplified by the Certal Residual DNA Assay Verification that the Certal Residual DNA Assay is working correctly in the case where no residual host-cell DNA is detected in the sample
50x Certal Internal Control Assay Predesigned, optimized, and validated primer-probe set (MAX labeled) for Certal Internal Control DNA detection Specific detection of Certal Internal Control DNA only. Optimized to prevent interference with Certal Residual DNA Assays
Certal Internal Control DNA Internal Control DNA that will be amplified by the Certal Internal Control Assay Excludes the presence of PCR inhibitors or can be used to control purification and amplification when added during the purification step
Certal ResDNA PCR Master Mix
HotStarTaq DNA Polymerase    15 min activation at 95°C Set-up of qPCR reactions at room temperature
Certal ResDNA PCR Buffer A balanced combination of KCl and (NH4)2SO4 ions Specific primer annealing ensures reliable PCR results
Synthetic Factor MP Reliable multiplexing analysis of up to 4 genes in the same tube
dNTP mix Includes dUTP, which partially replaces dTTP and enables optional UNG treatment Effective elimination of carryover of PCR products when the reaction is pretreated with uracil-N-glycosylase (UNG)
Additional components
50x 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
50x 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
Uracil-N-Glycosylase (UNG) Separate tube of pure, nuclease-free recombinant enzyme Effective elimination of carryover of PCR products
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
The Certal Residual DNA Detection Kit also includes Buffer TE for reconsititution of the lyophylized assays and RNase-free water.
操作手順

Certal Residual DNA Detection Kits provide a simple procedure for the detection of host-cell DNA, the Certal Internal Control, and the Certal Residual DNA Positive Control (see figure Certal Residual DNA Detection Kit procedure). Simply mix the supplied master mix with the Certal Residual DNA Assay, the Certal Internal Control Assay and the Certal Internal Control DNA, and then add your sample DNA or Certal Residual DNA Positive Control. The kit handbook contains optimized protocols for use on a wide range of cyclers. ROX is supplied in 2 tubes of different concentrations, enabling use on any real-time instrument (see table "Choosing the correct ROX concentration").

Choosing the correct ROX concentration
ROX Dye Solution
Instrument Not required ROX Dye Solution High-ROX Dye Solution
Rotor-Gene Q X
Rotor-Gene 6000 and Rotor-Gene 3000 X
Applied Biosystems 7500, ABI ViiA7 X
Applied Biosystems 7900HT, StepOne and StepOne Plus, ABI PRISM 7700, 7000 and 7300 X
Mx3000P, Mx3005P, Mx4000 X Optional
iCycler iQ System X
LightCycler 480 X
アプリケーション

Certal Residual DNA Detection Kits enable sensitive real-time PCR using sequence-specific probes for the detection of host-cell DNA, 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, and Agilent.

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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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Reliable detection of the QIAGEN Internal Control
Certal Residual DNA Detection Kit操作手順

ターゲット(CHO)アッセイ用のプライマーとプローブとインターナルコントロールをCertal ResDNA Master Mixに添加した。インターナルコントロールおよびサンプルのテンプレートを添加し、反応溶液を分注すれば、様々なサイクラーで反応を開始できる。オプションでポジティブCHOコントロールがテストと、PCR産物の混入をコントロールするためにUNGを使用もできる。

Certal Residual DNA Detection Kit procedure
Certal Residual DNA Detection Kit procedure.
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.
Sensitive and reliable detection of CHO genomic DNA on the Rotor-Gene Q

Rotor-Gene QでCHOゲノムDNAの感度と信頼性の高い検出

Rotor-Gene QでのCertal Residual DNA Assayの信頼性の高いことが、ターゲットシグナルの増幅プロットが等間隔であること、およびインターナルコントロールの安定したCT値により証明され(挿入図)、インターナルコントロールの高感度検出がCHOゲノムDNAに影響されないことを示す。
Low limit of detection

低い検出限界

24 replicatesで100% ポジティブな増幅結果が得られる最少濃度として定義された検出限界(LOD)は、Certal CHO Detection Kit Assayを使用して1 fgであった。標準値の±0.5 CT 内にある未知数の計算値の最少濃度として定義された定量限界(LOQ)は10 fgである。
Sensitive and reliable detection on a wide range of cyclers

様々なサイクラーで感度と信頼性の高い検出

3 fgから30 ngのターゲットDNAを、Certal CHO Detection Kitを用いて汎用されている様々なサイクラーで解析した。広範囲にわたる検出の直線性は、いくつかの異なるqPCRサイクラーでCertal アッセイが信頼性と適応性のあることを証明した。

LG_0187_Certal

感度および信頼性の高い検出

[A] Vero DNA(0.3 fg ~30 ng) あるいは [B] CHO DNA(0.3 fg~300 ng) を、相当するCertal Residual DNA Detection Kitを用いてApplied Biosystems ABI 7900 HT(384ウェルフォーマット)で解析した。高感度検出が低い定量限界と広い直線性の検出範囲で可能であった:[A] 3 fgから30 ngおよび [B] 3 fgから300 ng。反応の信頼性を示すエラーバーは4 replicates実験の±1 SDを示しているが、データポイントよりも小さいエラーバーは表示されていない。 LOQ:標準値の±0.5 CT内にある未知数の計算値の最少濃度として定義された定量限界。これは実験的に決められた:未知数の平均値は標準曲線の平均よりも0.5 CT 以上ずれていなかった。 LOD:検出限界、24 replicatesで100% ポジティブな増幅結果が得られる最少濃度として定義される。