0 item(s)
View cart
You have no items in your shopping cart.
Product Description
SHENTEK® rcAAV Quantitation Kit is a qPCR-based kit for detection and quantitation of replication-competent adeno-associated virus (rcAAV) contamination in recombinant AAV (rAAV) serotypes designated as rAAV-2/N. The kit is designed to quantify rcAAV-2/N contamination in rAAV production samples including harvested cell-culture bulk, purified stocks and final products. The kit contains a T&R DNA control for preparation of an absolute quantitation standard curve, separate Target and Reference Primer & Probe mixes, qPCR reaction buffer and supporting reagents. The kit supports standard-curve (absolute) quantitation and is compatible with common qPCR platforms (e.g., ABI 7500, Bio-Rad CFX96, Roche 480, SHENTEK-96S).
Important: The ITR sequence of the rAAV test sample must match the provided ITR sequence for correct detection. Reagents supplied are sufficient for 2 × 100 reactions.
Technical Specifications
| Parameter | Details |
|---|---|
| Intended use | Quantitative detection of replication-competent AAV (rcAAV-2/N) contamination in rAAV-2/N samples by qPCR (absolute quantitation, standard curve) |
| Number of reactions (kit size) | Reagents for 2 × 100 reactions |
| Dynamic range / standard curve | 20 to 2 × 10^6 copies/µL (standard curve concentrations: ST6=2×10^1 to ST1=2×10^6 copies/µL) |
| Limit of quantification (LOQ) | 20 copies/µL (lowest standard used for quantitation) |
| Reaction volume | 30 µL total reaction volume (10–10.04 µL qPCR mix + 20 µL sample/standard per well) |
| Thermal cycling (example ABI 7500) | Pre-melt 95 °C 10:00 (1 cycle); Denature 95 °C 00:15; Anneal/extend 60 °C 00:30; Read at 72 °C 00:30; 40 cycles. (Note: ABI 7500 users set final read step to 72 °C 34 s.) |
| Detector channels / probes | Target-2 detected in FAM channel; Reference-2 detected in CY5 channel; separate primer/probe mixes provided |
| Ct threshold settings (recommended) | Reference-2 threshold = 0.06; Target-2 threshold = 0.02; use automatic baseline and manual Ct |
| Ct acceptance criteria | NTC Ct (Reference and Target) should be >35 or undetermined. NCS Ct values should be larger than mean Ct of lowest standard. |
| Contamination calculation | rcAAV contamination rate = (Detection value of Target genes) ÷ (Detection value of Reference genes × dilution factor ÷ 2). Note: each rAAV contains two copies of the Reference gene. |
| Compatible instruments | ABI 7500, Bio-Rad CFX96, SHENTEK-96S, Roche 480 and other real-time PCR systems (ROX usage depends on instrument) |
| Storage stability | Kit components can be stored under appropriate conditions for up to 24 months |
Features
- Designed for rcAAV-2/N detection: Specifically optimized to quantify replication-competent AAV contamination in rAAV-2/N serotypes using ITR-targeted assays.
- Absolute quantitation by standard curve: Includes T&R DNA Control and dilution scheme to generate a 6-point standard curve (2×10^1 – 2×10^6 copies/µL) for absolute quantitation.
- Complete assay components: Provides qPCR reaction buffer, Target and Reference primer/probe mixes, ROX option, DNA dilution buffer and DNA control for streamlined setup.
- Platform flexibility: Compatible with multiple real-time PCR instruments; ROX addition is optional depending on instrument model.
- Procedural controls and guidance: Includes NTC and NCS guidance, recommended plate layouts, Ct thresholds, and sample dilution recommendations to support robust QC workflows.
Applications
- Quantitation of rcAAV contamination in recombinant AAV production (rAAV stocks and final products)
- Quality control testing during rAAV manufacturing
- Analysis of harvested cell-culture samples for presence of replication-competent AAV
- Lot release and batch testing for rcAAV contamination
Kit Contents
T&R DNA Control-2 (lyophilized powder, 1 tube)-20 °C
rcAAV qPCR Reaction Buffer (400 µL × 4 tubes)-20 °C (protect from light)
Target Primer & Probe MIX-2 (200 µL × 1 tube)-20 °C (recommended)
Reference Primer & Probe MIX-2 (200 µL × 1 tube)-20 °C (recommended)
100× ROX (20 µL × 1 tube)-20 °C (recommended)
DNA Dilution Buffer (DDB) (1.5 mL × 4 tubes)-20 °C
ddH2O (1 mL × 1 tube)2–8 °C (or -20 °C long-term)
* Total: 2 × 100 reactions | Method: qPCR (Absolute Quantitation by Standard Curve)
Attention
• For Research Use Only (RUO). Not for use in diagnostic procedures.
• Read Material Safety Data Sheets (MSDS) and wear appropriate PPE (gloves, lab coat, eye protection, mask) when handling reagents.
• Prevent nucleic acid contamination: UV-treat surfaces, pipettes and tubes for 30 minutes and disinfect with 75% alcohol before setup.
• Benzonase nuclease treatment of rAAV stocks prior to extraction is required to remove non-encapsidated nucleic acids and improve direct-qPCR accuracy.
• Follow the SHENTEK Virus DNA & RNA Extraction Kit instructions for sample extraction when applicable.
• Thaw reagents at 2–8 °C or on ice, vortex and briefly centrifuge before use; protect light-sensitive reagents (reaction buffer) from light.
• Centrifuge the lyophilized DNA control at 12,000 rpm for 1 min and reconstitute precisely with 55 µL ddH2O before preparing dilutions.
• Prepare standard curve fresh (recommended six points) and run samples in triplicate for reliable quantitation.
• Add 100× ROX only if required by the qPCR instrument (ABI 7500 requires ROX; SHENTEK-96S and Roche 480 generally do not).
• Use recommended Ct threshold settings (Reference = 0.06; Target = 0.02) and check slope/intercept/R² of standard curve for assay validity.
Quality Management & Certifications
Quality System
Download QMSQMS (ISO, GMP)
Download CertificateQuality Advantages
View ReportQuality Control Process
Download ProcessTechnical Resources & Downloads
Frequently Asked Questions (FAQ)
Q1: What sample types can be tested with this kit?
This kit supports purified rAAV bulk, final product stocks and harvested cell-culture samples. For bulk or final product stocks, perform Benzonase treatment and nucleic acid extraction prior to qPCR. Cell culture samples may be extracted using the SHENTEK Virus DNA & RNA Extraction Kit.
Q2: What is the assay dynamic range and lowest quantifiable concentration?
The standard curve covers 2 × 10^1 to 2 × 10^6 copies/µL (20 to 2,000,000 copies/µL). The lowest standard used for quantitation (LOQ) is 20 copies/µL.
Q3: How is rcAAV contamination rate calculated?
rcAAV contamination rate = (Measured copies of Target gene) ÷ (Measured copies of Reference gene × dilution factor ÷ 2). Note that each rAAV contains two copies of the Reference gene.
Q4: Do I need to add ROX to every instrument?
No. Addition of 100× ROX is instrument-dependent. ABI 7500 requires 0.04 µL ROX per reaction for the Target assay; SHENTEK-96S, Roche 480 and some other systems do not require ROX. Follow your instrument documentation.
Research Use Only
✔
Research Use Only (RUO). For Research Use Only. Not for use in diagnostic procedures. – This product is intended for laboratory research purposes only and not for clinical or regulatory diagnostic use.
✔
Not intended for use in USDA or FDA regulated diagnostic testing or official compliance testing.
When can I expect my order to ship?
Most orders are filled and shipped within 2-3 business days from the time they are received.
Our standard shipping usually take 2-5 days.
We also provide express shippping for time-sensitive deliveries.
Email contact@biofargo.com if you have any requirements.
