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Product Description
SHENTEK® rcAAV Quantitation Kit is designed for qPCR detection and absolute quantitation of replication-competent adeno-associated virus (rcAAV) contamination in rAAV-5/N serotypes. The kit is suitable for testing recombinant AAV bulk, purified end-products and harvested cell-culture samples. The assay targets the ITR sequence of rAAV-5/N and includes T&R DNA control material, separate Reference and Target primer/probe mixes, and reagents for construction of a standard curve for absolute quantitation. Benzonase treatment of rAAV prior to extraction is recommended to remove unprotected nucleic acids and improve accuracy of direct qPCR measurements.
Technical Specifications
| Parameter | Details |
|---|---|
| Standard curve range (linear range) | 20 - 2 × 10^6 copies/µL |
| Standard points (example) | ST1 = 2 × 10^6 copies/µL; ST2 = 2 × 10^5; ST3 = 2 × 10^4; ST4 = 2 × 10^3; ST5 = 2 × 10^2; ST6 = 2 × 10^1 |
| Reaction volume | 30 µL total reaction volume (10 µL qPCR MIX + 20 µL sample/standard/DDB per well) |
| qPCR MIX composition (per reaction) | Reference: 8 µL rcAAV qPCR Reaction Buffer + 2 µL Reference Primer&Probe MIX-5; Target: 8 µL rcAAV qPCR Reaction Buffer + 2 µL Target Primer&Probe MIX-5 (+ 0.04 µL 100×ROX optional for some instruments) |
| Thermal cycling program (example / ABI7500) | Pre-melt 95 °C 10:00 (1 cycle); Activation 95 °C 0:15; Denature 60 °C 0:30; Anneal/Extend 72 °C 0:30 (read fluorescence during this step); 40 cycles. (Note: ABI7500 users: change final step timing to 72 °C 34 s.) |
| Instrument compatibility | ABI 7500, Bio-Rad CFX96, SHENTEK-96S, Roche LightCycler 480 (and other real-time PCR instruments with appropriate dye settings) |
| Threshold settings (recommended) | Reference-5 threshold = 0.06; Target-5 threshold = 0.02; Manual Ct, Automatic baseline |
| NTC / NCS acceptance criteria | Ct (Reference and Target) for NTC should be >35.00 or undetermined. Ct values for NCS should be larger than mean Ct of lowest standard. |
| Replicates | Triplicates recommended for standards, controls and samples |
| Calculation of rcAAV contamination rate | Contamination rate = (Detection value of Target genes) ÷ (Detection value of Reference genes × dilution factor ÷ 2). Note: each rAAV contains two copies of Reference gene. |
Features
- rcAAV-specific detection: Designed to detect replication-competent AAV contamination (rcAAV) in rAAV-5/N serotypes by targeting the ITR sequence.
- Absolute quantitation: Includes a T&R DNA control and instructions for preparing a 6-point standard curve enabling absolute quantitation (copies/µL).
- Ready-to-use primer/probe mixes: Separate Reference and Target Primer&Probe MIX-5 provided to streamline assay setup.
- Instrument flexibility: Compatible with multiple real-time PCR platforms (ABI 7500, CFX96, SHENTEK-96S, Roche 480) with guidance for ROX usage per instrument.
- Quality control guidance: Includes recommended controls (NTC, NCS) and acceptance criteria (Ct thresholds) to support assay validity.
Applications
- Quantification of rcAAV contamination in rAAV-5/N production lots
- Quality control testing of rAAV bulk and purified end-products
- Testing harvested cell-culture samples for rcAAV following infection/production
- In-process monitoring during rAAV manufacturing and downstream purification
Kit Contents
T&R DNA Control-5 (NNA028)-20 °C (lyophilized)
rcAAV qPCR Reaction Buffer (NNB009) 400 µL × 4 tubes-20 °C, protect from light
Target Primer&Probe MIX-5 (NNC050) 200 µL × 1 tubeRefer to manual
Reference Primer&Probe MIX-5 (NNC051) 200 µL × 1 tubeRefer to manual
100× ROX (NND007) 20 µL × 1 tubeRefer to manual
DNA Dilution Buffer (DDB) (NND001) 1.5 mL × 4 tubes-20 °C
ddH2O (NND010) 1 mL × 1 tubeRefer to manual
* Total: Reagents for 2 × 100 reactions | Method: qPCR (absolute quantitation / standard curve)
Attention
• For research use only. Read Material Safety Data Sheets (MSDS) and follow handling instructions; wear appropriate PPE (gloves, eyewear, mask, lab coat).
• Benzonase nuclease treatment of rAAV stock or final product is recommended prior to nucleic acid extraction to remove unprotected nucleic acids and improve accuracy of direct qPCR.
• Work area, pipettes and tubes should be decontaminated (UV 30 min, 75% ethanol) before setup to avoid contamination.
• Thaw reagents at 2–8 °C or on ice; vortex and briefly centrifuge prior to use. Reconstitute lyophilized DNA control with precise volume and ensure complete dissolution (centrifuge and invert as instructed).
• Store kit components under recommended conditions and check expiration date. Unused DDB should be stored at 2–8 °C; if cloudy, warm to 37 °C until clear.
• Include at least five concentration points in the standard curve; perform triplicates for standards, controls and samples.
• Ensure sample dilutions place sample within the standard curve range (20–2 × 10^6 copies/µL) for Reference gene. Target gene may be tested undiluted for high-titer samples; dilute Reference measurements as needed.
• Set instrument-specific ROX usage according to instructions (e.g., ABI7500: add 0.04 µL ROX per reaction; other instruments may not require ROX).
• NTC Ct values should be >35 or undetermined; NCS Ct must be larger than mean Ct of lowest standard. Multiply measured concentration by sample dilution factors for final copies/mL reporting.
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 is the quantitation range of the kit?
The validated standard curve range is 20 to 2 × 10^6 copies/µL (use ST1–ST6 standards as described).
Q2: How should I store the kit and reagents?
Store kit components at recommended temperatures; frozen components are stored at -20 °C. Check individual component labels and the user guide for storage and stability (kit stable for up to 24 months).
Q3: What controls should be included with each run?
Include a no-template control (NTC), a negative quality control (NCS), and a standard curve (minimum five concentrations recommended). Test samples and controls should be run in triplicate.
Q4: How is rcAAV contamination rate calculated?
Contamination rate = (Target gene copies) ÷ (Reference gene copies × dilution factor ÷ 2). Note that each rAAV contains two copies of the Reference gene.
Research Use Only
✔
Research Use Only (RUO) – 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.
