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.

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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
211822
1.380,00 €

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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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 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").

Principle

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.
Procedure

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
Applications

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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Kit Handbooks
1
For sensitive real-time PCR detection of residual host cell DNA and an internal control using sequence-specific probes
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