| Field | Specification |
|---|---|
| Product Type | |
| Shipping | |
| Storage |
Description
The RCA (E1A) Copynumber Detection Kit can be used to quantitatively analyze and detect the potential risk of Replication-competent Adenoviruses (RCA) that may occur in a variety of cellular therapeutic products and gene therapy products associated with the use of adenoviral vectors; and can also be used to quantitatively analyze and detect the presence of residual DNA of the risk gene E1A in biologics from host cells, such as HEK293 and HEK293T cell-derived risk gene E1A residual DNA.
This kit designs specific primers for the E1A gene sequence on adenovirus, and adopts the principle of fluorescence quantitative PCR by probe method to rapidly and specifically detect the risk of replicative adenovirus RCA and the residual DNA of E1A from the host cell, and the limit of quantification can be as low as 4.1 copies/μL, and it contains the RCA (E1A) DNA Control (DNA quantitative reference product). The kit needs to be used together with the the Residual DNA Sample Preparation Kit (Cat# 18461ES).
Feature
- Compliance with regulations: the products are fully validated according to the requirements of Chp, USP, ICH, etc., and their performance is in line with Chinese and foreign regulations and standards;
- Guarantee of quality: the raw materials of the kits are all self-developed, and the qPCR Mix and other enzyme products are produced in an ultra-clean enzyme factory;
- High sensitivity: the lower limit of quantification (LLOQ) was 4.1 copies/uL;
- High precision: high intra-batch reproducibility CV <10%, small inter-batch variation i.e. intermediate precision CV <15%;
- Strong specificity: specific detection of RCA (E1A) residual DNA without interference from other exogenous genomic DNA;
- Strong anti-interference: the introduction of internal quality control (IC) can exclude sample interference, abnormal reaction preparation and other factors, effectively avoiding false negatives.
Application
- Residual RCA (E1A) DNA test in biological products.
Specifications
|
Sensitivity |
4.1 copies/uL (range 1.64×101～1.64×105copies/μL）) |
|
Assay Time |
~1.5 hours |
|
Assay Principle |
Fluorescent probe qPCR method |
|
Required Instruments |
Real-time fluorescence quantitative PCR system |
Product Performance
|
Product Performance Validation Program |
Reference standards or requirements |
YEASEN |
|
|
1. Standards for the kit |
Homemade standards are available |
Plasmid DNA containing Adenovirus E1A gene |
|
|
2. Linear Range |
Assay Range |
Refer to the actual situation |
1.64x101~1.64x105 copies/μL |
|
R2 |
≥0.98 |
≥0.99 and above |
|
|
Slope |
-3.1~-3.8 |
-3.1~-3.6 |
|
|
Amplification Efficiency |
84%~109% |
90%~109% |
|
|
3. Accuracy |
Recovery Rate |
50%~150% |
70%~130% and above |
|
4. Precision |
Repeatability |
CV≤15% |
＜10% |
|
Intermediate precision |
CV≤15% |
＜15% |
|
|
5. Exclusivity |
No interference with DNA from other sources |
Specific detection of RCA DNA without interference from other exogenous DNA |
|
|
6. Sensitivity |
Limit of quantification |
Reliable quantification of the smallest concentrations of target substances |
4.1 copies/μL |
|
7. Durability |
Reagent Stability |
Evaluating the effect of different storage temperatures on reagent performance |
4°C for 14 days and 37°C for 14 days, and repeated freeze-thaw 10 times, the kit amplification efficiency is in the range of 90%~109%, R2≥0.99 and above |
|
Instrument Compatibility |
Kits are used on different devices without affecting the test results |
Compatible with many types of qPCR Instruments (eg. Bio-Rad CFX96 Optic Module, Thermo Scientific ABI 7500, ABI Quant Studio 5) |
|
Components
|
Components No. |
Name |
41321ES50 |
41321ES60 |
|
41321-A |
RCA (E1A) qPCR Mix |
0.75 mL |
1.5 mL |
|
41321-B |
RCA (E1A) Primer&Probe Mix |
200 μL |
400 μL |
|
41321-C |
DNA Dilution Buffer |
1.8 mL×2 |
1.8 mL×4 |
|
41321-D |
RCA (E1A) DNA Control (1.64×107copies/μL) |
25 μL |
50 μL |
|
41321-E |
IC* |
50 μL |
100 μL |
*IC：Internal control.
Storage
This product should be stored at -25~-15℃ for 2 years.
Both 41321-A and 41321-B should be stored protected from light.
Figures
- Limit of quantification
RCA DNA at concentrations of 1.64×101copies/uL, 8.2copies/uL, 4.1copies/μL, 2.05copies/μL and 1.64copies/uL were detected, with 10 replicates for each concentration, respectively. The results showed that the CV of RCA DNA was <20% at concentrations of 4.1copies/μL and above. Therefore, the limit of quantification in the RCA DNA residue detection kits was 4.1copies/μL for RCA.
Table 1. Limit of quantification (LOQ) assay results for RCA DNA
|
Repetition number
Test item |
RCA (copies/μL) |
|
|
Quantity |
Return ratio |
|
|
1 |
3.77 |
91.95% |
|
2 |
4.44 |
108.29% |
|
3 |
3.75 |
91.46% |
|
4 |
4.31 |
105.12% |
|
5 |
4.00 |
97.56% |
|
6 |
4.50 |
109.76% |
|
7 |
3.56 |
86.83% |
|
8 |
3.55 |
86.59% |
|
9 |
3.59 |
87.56% |
|
10 |
3.66 |
89.27% |
|
Average value |
3.91 |
\ |
|
CV |
9.57% |
\ |
Figure 1. 4.1copies/μL RCA (E1A) DNA qPCR assay results
Documents:
Safety Data Sheet
Manuals
Host cell DNA (HCD) residue testing is required for biopharmaceutical manufacturing under ICH Q5A, ICH Q6B, FDA 21 CFR Part 610, and EMA guidelines. Acceptable residual HCD limits are typically ≤10 ng/dose (WHO guideline) or ≤100 pg/dose for continuous cell line-derived products, depending on the product class and route of administration. This kit provides qPCR-based quantification traceable to validated reference standards.
This kit uses primers and probes targeting genome-specific repetitive elements (e.g., Alu elements for human HEK293/CHO, LINE-1 for mammalian cells, or species-specific rDNA repeats for yeast/insect cell lines) that are highly conserved and abundant in the host genome. This provides high sensitivity — typically detecting sub-picogram quantities of residual host DNA — without cross-reacting with the target product or plasmid sequences.
Yes. The quantitative qPCR format of this kit is suitable for lot release testing, process characterization, and stability monitoring of biopharmaceutical drug substances and drug products. To generate regulatory-compliant data, assay performance qualification (sensitivity, specificity, linearity, spike recovery) should be documented per applicable guidelines. Contact your QA team to confirm kit suitability for your specific regulatory submission.
The LOD varies by kit generation and target cell line (e.g., HEK293, CHO, E. coli, Pichia pastoris). Third-generation (3G) kits typically achieve LOD values of 0.1–1 pg of host cell DNA per reaction. The validated linear range spans 4–6 orders of magnitude, supporting both low-level process monitoring and high-level spiking experiments. Validated LOD and LOQ values are provided in the product-specific datasheet.
Yeasen Biotechnology offers flexible customization options for many of its assay kits and detection reagents, including custom lot sizes, bulk ordering, and application-specific formulation adjustments. Volume pricing, custom packaging, and kit bundling may be available depending on the product and intended workflow. A Certificate of Analysis (CoA) and lot-specific QC data are provided with every order. For inquiries regarding large-volume orders, custom configurations, or integration into automated workflows, please contact the BioHippo team for a tailored quotation.
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