QIAcuity Residual DNA Quantification Kits

For checking carryover of host cell DNA with higher accuracy and precision using the QIAcuity Digital PCR System

S_1261_5_LS_dPCR_QIAcuity_eColi_resDNA_Quant

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

E. coli resDNA Quant Standard Kit

Cat. No. / ID:   250221

E. coli resDNA Quant Standard (1x), Rehydration Buffer
Kit
resDNA Quant Standard
resDNA Quant
Cell type
E. coli
CHO
HEK293
QIAcuity Residual DNA Quantification Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • Premixed master mix with controls for easy set up and detection of host cell DNA
  • Enables accurate detection of residual host-cell DNA from E.coli, CHO and HEK293 cells down to low femtogram levels
  • A multi-copy target assay that detects and quantifies highly fragmented host cell DNA
  • Enables host cell DNA detection in both extracted and unextracted samples

Product Details

Host cell DNA contamination in products such as vaccines, drugs and other biopharmaceuticals imposes great health risks. Therefore, its safe limits are tightly regulated by agencies such as the U.S. FDA and WHO. Clear guidelines for the upper limits of residual DNA are established based on the nature of drug administration, infectivity and oncogenicity of the contaminating cell DNA. For instance, parenteral administration of non-tumorigenic cell DNA should be limited to 10 ng/dose and a maximum length of 200 bp, whereas less than 100 µg/dose of residual DNA is recommended by WHO for orally administered vaccines. 


Detection and removal of such contaminations in manufacturing products require highly sensitive and accurate measurements of the extremely low amounts of specific host cell DNA present in the products.


Digital PCR is the preferred method of detection for residual DNA quantification. This is due to its unmatched sensitivity and accuracy, especially at trace levels of contamination compared to other detection methods such as qPCR. QIAcuity Residual DNA Quantification Kits provide the accurate and precise detection of Escherichia coli (E.coli), Chinese Hamster Ovary cell (CHO) and Human Embryonic Kidney 293 cells (HEK293) host cell DNA.     

 

Performance

The QIAcuity Residual DNA Quantification Kits provide accurate CHO, E.coli and HEK293 residual DNA (resDNA) quantification results with or without extraction, even in the presence of PCR contaminants or other inhibitory reagents. The assays are multicopy target assays that accurately determine highly fragmented residual host cell DNA.

  

Assay Target Copy Number Amplicon Size Conversion Factor cp/µl to fg/µl
QIAcuity E.coli resDNA Quant Kit 7 <200 0.35
QIAcuity CHO resDNA Quant Kit ~ 1 million, undefined (repeated element) <100 0.28
QIAcuity HEK293 resDNA Quant Kit ~ 1 million, undefined (repeated element) <100 1.54

 

QIAcuity E.coli resDNA Quant Kit detects as low as 5 fg of residual DNA in a single reaction

Loading amount per reaction (fg/rxn) E.coli Standard (copies/µl) Internal Control (copies/µl)*
50000 2949.7 99.9
5000 291.6 91.7
500 27.6 91.4
50 2.8 93.2
25 1.46 92.5
5 0.45 90.6
NTC 0 98.9

*100 copies/µl expected for Internal Control

Digital PCR provides higher sensitivity of detection at a lower template input range in comparison to qPCR and therefore enables a more robust application.

QIAcuity CHO resDNA Quant Kit detects as low as 5 fg of residual DNA in a single reaction

Loading amount per reaction (fg/rxn) CHO Standard (copies/µl) Internal Control (copies/µl)*
50000 4939.3     108.7
5000 508.3     104.9
500 50.2     97.6
50 4.7 99.5
25 2.6 101.3
5 0.6 100.4
NTC 0 104.7

*100 copies/µl expected for Internal Control

QIAcuity HEK293 resDNA Quant Kit detects as low as 5 fg of residual DNA in a single reaction

Loading amount per reaction (fg/rxn) HEK293 Standard (copies/µl) Internal Control (copies/µl)*
50000 1104.9 96.6
5000 104.5 92.5
500 8.83 92.4
50 0.99 94.4
NTC 0 96.2

*100 copies/µl expected for Internal Control

The kits work in conjunction with the QIAcuity Digital PCR System and QIAcuity Nanoplates, offering an end-to-end fast dPCR workflow comparable to qPCR, but delivering absolute quantification of resDNA in your sample. The kits have been designed with the requirements of bioprocess manufacturing and QC in mind.

 

Principle

The principle of the dPCR reaction in the nanoplates is described here.

Using QIAcuity digital PCR for host cell DNA monitoring improves the LOD/LOQ through partitioning of the bulk sample with the help of QIAcuity Nanoplate 26k. Partitioning increases the effective concentration of the target, allowing a small amount of host cell DNA to be captured and measured with higher accuracy and precision. The ability to load high template input amounts in the QIAcuity Nanoplate 26k combined with an increased number of partitions is one of the reasons for the higher accuracy and precision of residual host cell detection.

  

 

Procedure

The lysate is added to the residual quant assay together with the internal control and host cell DNA measured by absolute quantification. Copies/µl is converted to fg/µl via a provided conversion factor.

Applications

The QIAcuity Residual DNA Quantification Kits are ideal for the highly precise quantification of host cell DNA in complex bioprocess intermediates.

Supporting data and figures

FAQ

What does the standard kit contain?
The standard for HEK293, CHO, and E. Coli contains genomic DNA, and we recommend digestion of the standard for better migration out into the partitions.
FAQ ID - 3938
dPCR results are in copies per microliter. How do I calculate host cell DNA contamination in femtogram per microliter?

The conversion factors from copies per microliter to femtogram per microliter for each target assay (E. coli, CHO, and HEK293) will be provided in the quick-start protocol and the QIAcuity User Manual Extension: Application Guide.

FAQ ID - 3876
My samples are highly fragmented. Is the dPCR result for resDNA quantification accurate?

ResDNA Quant Kit (E. coli, CHO, and HEK293) assays are multicopy target assays that ensure quantification of  resDNA unaffected by the fragmentation level. In addition, short amplicon regions are selected for each target region. 

FAQ ID - 3877
Is DNA extraction from the samples needed?

No. DNA extraction is not required. However, if the samples contain high levels of PCR inhibitors or high protein concentration, DNA extraction is recommended for accurate quantification of ResDNA contamination.

FAQ ID - 3873
What is the amount of standard for the different kit?
The standard that is provided has an amount of 1000 pg that will be diluted with 100 µL of rehydration buffer down to working solution of 10 pg/µL.
FAQ ID - 3936
Is restriction enzyme digestion required?

Restriction digestion of the templates is required when the template length exceeds 20 kb for a better distribution of templates within partitions. Recommended restriction enzymes can be found in the applications guide and quick-start protocols.

FAQ ID - 3875
Which extraction kits are recommended?

QIAamp UCP DNA Micro Kits, QIAamp HMW MagAttract Kits, QIAsymhony DSP Kits, QIAsymphony Certal Kits, and DNeasy Blood & Tissue Kits are few recommended extraction kits to combine with ResDNA Quant Kit.

FAQ ID - 3874
What is the preferred storage temperature for reconstituted assay for the QIAcuity HEK293 resDNA Quant Kit (96)?
The recommended storage temperature is 4°C for 72 hours.
FAQ ID - 3935
Is dPCR sensitive to inhibition?

dPCR is less prone to inhibition in comparison to qPCR; however, we recommend dilution of samples that are known to contain PCR inhibitors.

FAQ ID - 3880
How much sample can I load for detecting host cell contamination?

26k Nanoplates are recommended for resDNA quantification, which maximizes the amount of template that can be loaded within a single reaction. In 26k nanoplates, this corresponds to 28 μl in 40 μl reaction volume. In addition, maximum template loading amounts for each kit will be provided in QSPs. 

FAQ ID - 3879
What are the ResDNA Quant Kit components?

The kits are composed of a premixed master mix for CHO and E. coli in liquid format that contains the target. For HEK293 the same composition comes in lyophilized format. The kit also contains an internal control assay detected in Green and Yellow channel, internal control DNA, positive control DNA, and dPCR qualified water. All necessary components are included in the ResDNA Quant Kits.

FAQ ID - 3878
How long can I store the reconsituted assay regarding QIAcuity HEK293 resDNA Quant Kit (96) at 4°C?
For optimal performance, the recommendation is to use the reconstituted assay within 72 hours.
FAQ ID - 3934
What region is targeted in the QIAcuity E. coli resDNA Quant Kit (96)?
The QIAcuity E. coli resDNA Quant Kit (96) is targeting the transcribed genomic regeion of 16S with <200 bp fragment size. 
FAQ ID - 3939
How did you calculate the conversion factor?
A serial dilutions of HEK293, CHO, and E. Coli gDNA standard test them using HEK293, CHO, and E. Coli ResDNA Quant Kits, respectively. We then compare the copies per microliter obtained at different loading concentrations. We correlate the number of copies of target amplicon with the loading amount in picogram. To rule out any issues with PCR performance, loss of target amplicons, loading errors, user error, instrument errors, etc. skewing the true conversion factor, we repeat the testing over multiple samples, multiple batches of kits, standards, and multiple users. This gives us our accurate conversion factor.
FAQ ID - 3937