| Field | Specification |
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Description
The Replication-competent Retrovirus (RCR) Detection Kit can be used to quantitatively analyze and detect the potential risk of Replication-competent Retrovirus (RCR) that may occur in a variety of cellular therapeutic products and gene therapy products associated with the use of retrovirus vectors.
This kit targets specific sequences on the retroviral vector to design specific primers, and adopts the principle of fluorescence quantitative PCR by probe method to rapidly and specifically detect the risk of replicative retrovirus (RCR). And the limit of quantification can be as low as 10 copies/μL, the limit of detection can be 0.3 copies/μL, it contains the RCR DNA Control (DNA quantitative reference product). The kit needs to be used together with the the Residual DNA Sample Preparation Kit (Cat# 18461ES).
Features
- 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 10 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 RCR 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 RCR DNA test in biological products.
Specifications
|
Sensitivity |
10 copies/uL (range 5×101～5×106copies/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 |
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1. Standards for the kit |
Homemade standards are available |
Plasmid DNA containing specific sequences on the retroviral vector |
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2. Linear Range |
Assay Range |
Refer to the actual situation |
5x101~5x106 copies/μL |
|
R2 |
≥0.98 |
≥0.99 and above |
|
|
Slope |
-3.1~-3.8 |
-3.1~-3.6 |
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|
Amplification Efficiency |
84%~109% |
90%~109% |
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|
3. Accuracy |
Recovery Rate |
50%~150% |
70%~130% and above |
|
4. Precision |
Repeatability |
CV≤15% |
＜10% |
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Intermediate precision |
CV≤15% |
＜15% |
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|
5. Exclusivity |
No interference with DNA from other sources |
Specific detection of RCR DNA without interference from other exogenous DNA |
|
|
6. Sensitivity |
Limit of quantification |
Reliable quantification of the smallest concentrations of target substances |
10 copies/μL |
|
Limit of detection |
Refer to the actual situation |
0.3 copies/μL |
|
|
7. Durability |
Reagent Stability |
Evaluating the effect of different storage temperatures on reagent performance |
2~8°C for 30 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 |
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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) |
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Components
|
Components No. |
Name |
41329ES50 |
41329ES60 |
|
41329-A |
RCR RT-qPCR Mix (Primer&Probe) |
850 μL |
1.7 mL |
|
41329-B |
RCR RT-qPCR Enzyme Mix |
100 μL |
200 μL |
|
41329-C |
Dilution Buffer |
1.8 mL×2 |
1.8 mL×4 |
|
41329-D |
RCR Control (5×108 copies/μL) |
25 μL |
50 μL |
|
41329-E |
RCR IC* |
50 μL |
100 μL |
*IC：Internal control.
Storage
This product should be stored at -25~-15℃ for 2 years.
The 41329-A should be stored protected from light.
Figures
- Limit of quantification
RCR DNA at concentrations of 5×101 copies/uL, 30 copies/uL, 20 copies/μL, 10copies/μL and 5 copies/uL were detected, with 10 replicates for each concentration, respectively. The results showed that the CV of RCR DNA was <20% at concentrations of 10 copies/μL and above. Therefore, the limit of quantification in the RCR DNA residue detection kits was 10 copies/μL for RCR.
Table 1. Limit of quantification (LOQ) assay results for RCR DNA
|
Repetition number
Test item |
RCR (copies/μL) |
|
|
Quantity |
Return ratio |
|
|
1 |
9.97 |
99.7% |
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2 |
9.47 |
94.7% |
|
3 |
8.80 |
87.9% |
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4 |
9.51 |
95.1% |
|
5 |
10.46 |
104.6% |
|
6 |
8.91 |
89.1% |
|
7 |
10.72 |
107.2% |
|
8 |
9.88 |
98.8% |
|
9 |
8.65 |
86.5% |
|
10 |
10.96 |
109.6% |
|
Average value |
9.73 |
\ |
|
CV |
8.34% |
\ |
Figure 1. 10 copies/μL RCR DNA qPCR assay results
- Limit of detection
The detection of RCR DNA at various concentration points under the quantitative limit, combined with the blank limit requirements, yields the detection limit of the reagent kit. The results show that at concentrations of 0.3 copies/μL and above, in 20 repeated wells, ≥19 wells are detected (i.e., the detection rate is ≥95%). Therefore, the detection limit of the Replication-competent Retrovirus (RCR) Detection Kit can reach 0.3 copies/μL.
Table 2. Limit of detection (LOD) assay results forRCR DNA
|
Ct value Theoretical concentration |
RCR DNA (copies/μL) |
||
|
0.3 |
0.2 |
0.1 |
|
|
Positive count / Total count |
19/20 |
17/20 |
11/20 |
|
Detection rate |
95% |
85% |
55% |
Documents:
Safety Data Sheet
Manuals
Replication-competent virus (RCV) — including RCL (lentivirus), RCR (retrovirus), and rcAAV — can form when recombination events during viral vector production reconstitute a replication-competent genome. Detection is mandatory for gene therapy product release under FDA (21 CFR 312.23), EMA, and ICH Q5A guidelines. Even trace amounts of RCV pose significant biosafety risks in clinical applications due to their potential to integrate and replicate in patient cells.
This qPCR-based kit achieves high sensitivity with an LOD typically in the range of 1–10 copies per reaction for RCL/RCR detection, and less than 10 copies per reaction for rcAAV2 detection. Specificity is confirmed against wild-type viral sequences not present in replication-deficient vector preparations. Refer to the product datasheet for validated LOD, LOQ, and specificity data established with certified reference materials.
This kit provides a research-grade (RUO) molecular tool for RCV detection with sensitivity appropriate for process development and QC screening. For GMP lot release submissions to regulatory agencies, full assay qualification (sensitivity, specificity, linearity, robustness) must be conducted per applicable guidelines (ICH Q2R1, USP <1033>). Consider this kit as a validated starting platform for developing a GMP-qualified method.
This kit is validated for purified nucleic acid extracted from viral vector preparations, cell culture supernatants, and drug substance/product samples. Pre-analytical steps (extraction method, input volume) should be optimized for your specific matrix. For complex matrices such as formulated drug products containing stabilizers or preservatives, spike-recovery experiments should confirm assay performance before routine use.
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.
