Certal Residual DNA Detection Kits

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

Features

  • 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
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Certal CHO Detection Kit (100)

Cat. No. / ID: 211822

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
€3,074.00
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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. See trademarks.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

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.

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").

See figures

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
See figures

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.

Supporting data and figures

Resources

Quick-Start Protocols (1)
Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.
Kit Handbooks (1)
For sensitive real-time PCR detection of residual host cell DNA and an internal control using sequence-specific probes

FAQ

Can the Certal CHO Positive Control DNA be used to create a qPCR standard for relative quantification?
For quantification, it is recommended that a gDNA standard from your production cell be prepared using, for example, the QIAamp DNA Mini Kit. The positive control provided with Certal Residual DNA Detection Kits is not suitable for use as a qPCR standard for relative quantification.
FAQ ID -2979
How many freeze-thaw cycles can the Internal Control supplied with Certal Residual DNA Detection Kits tolerate?

The Internal Control supplied with Certal Residual DNA Detection Kits can be freeze-thawed up to 6 times without a decrease in performance. However, great care should be taken to avoid inadvertently introducing RNases or DNases into the Internal Control template solutions. In general, in order to avoid repeated freeze-thaw cycles, we recommend preparing aliquots of the Internal Control template.

Note:  The Internal Control supplied with Certal Residual DNA Detection Kits cannot be ordered separately

FAQ ID -2975
On which cyclers can Certal Residual Detection Kits be used?
Certal Residual Detection Kits can be used on virtually any real-time cycler. The master mix supplied with the Kits does not contain ROX. Instead, 2 different ROX concentrations are supplied in separate tubes. “High-ROX Dye Solution” is for use with all Applied Biosystems cyclers except the Applied Biosystems 7500 Real-Time PCR Systems and the ViiA7 System, and “ROX Dye Solution” is for use with the Applied Biosystems 7500 Real-Time PCR Systems, the ViiA7 System, and, optionally, instruments from Agilent. Recommendations are provided in the handbook (page 11).
FAQ ID -2973
What is the label of the probe that is used in the Internal Control Assay to detect the Internal Control supplied with Certal Residual DNA Detection Kits?
The probe is labeled with the MAX-NHS ester (MAX) reporter dye, which has a spectrum equivalent to HEX, JOE, or VIC dyes. IowaBlack is used as the quencher dye. IowaBlack is a non-fluorescent quencher (dark quencher).
FAQ ID -2976
If a cycler from Applied Biosystems is used in combination with the Certal Residual DNA Detection Kit, should dye calibration for MAX be performed?

No, calibration is required if the instrument is already calibrated for VIC (which is normally the case). Simply create a new detector, for example, “MAX/IowaBlack”, and assign VIC as the reporter dye and “none” as the quencher (Table 3, page 17 of the Certal Residual DNA Detection Kit handbook describes the procedure in the footnote). 

MAX spectrum is nearly identical to VIC and the requirement for calibration has been thoroughly tested. There is no risk of crosstalk from the VIC channel to the FAM channel. However, crosstalk may occur from the FAM channel to the VIC channel if the pathogen target is present in very high copy numbers. Although this may slightly change the signal for the Internal Control, if a positive signal is detected in the FAM channel, then the Internal Control signal is no longer required.

FAQ ID -2977
On what cyclers have Certal Residual DNA Detection Kits been validated?
Certal Residual DNA Detection Kits have been validated on the Rotor-Gene Q and the Applied Biosystems 7900HT Fast Real-Time System.
FAQ ID -2978
Is there a risk of crosstalk from the FAM channel to the VIC channel if the residual DNA target is present in very high copy numbers?
Usually the amount of gDNA in process samples is low, for example, in the ng range or even lower. Therefore, no crosstalk is expected.
FAQ ID -2980
What is the nature and target of the Internal Control supplied with Certal Residual DNA Detection Kits?

The Internal Control supplied with Certal Residual DNA Detection Kits is a non-linearized plasmid (naked nucleic acid).

The Internal Control supplied with Certal Residual DNA Detection Kits has an artificial sequence that is not present in biological sample material.

FAQ ID -2974