Products
The Viral Vector Lysis Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Cell and Gene Therapy Viral Vector Lysis Kit (100 rxn)
Cat. No. / ID: 250272
The kit includes: CGT Sample Stabilizer, DNase I, Nuclease-Free Water, CGT Lysis buffer, CGT DNase I buffer and CGT Dilution Buffer enough to process 100 AAV samples
Cell and Gene Therapy Viral Vector Lysis Kit (1000 rxn)
Cat. No. / ID: 250273
The kit includes: CGT Sample Stabilizer, DNase I, Nuclease-Free Water, CGT Lysis buffer, CGT DNase I buffer and CGT Dilution Buffer enough to process 1000 AAV samples
Features
- One kit and one protocol solution for AAV lysis enables standardization and quality control (QC) of current workflows
- Consistent and reproducible measurement of viral titers for multiple serotypes (e.g., AAV serotypes)
- Complete workflow combined with QIAcuity Cell and Gene Therapy (CGT) dPCR Assays and accurate measurement of AAV titers on the QIAcuity Digital PCR System
Product Details
Adeno-associated virus is a widely used viral vector in gene therapy applications. However, the process of generation and purification of the viral vectors requires precise quality control to enable safe and reliable dosing during clinical studies or patient care. The ability to accurately quantify vector titers as well as determine contaminations is key to safe and effective AAV-based gene therapies.
With the introduction of the CGT Viral Vector Lysis Kit that includes reagents for either 100 or 1000 reactions, we now have an optimized and standardized workflow for viral vector lysis through to an accurate and precise way of determining the viral titer. The kit is also compatible with adenoviruses.
Performance
We offer a complete workflow from performing AAV lysis to quantification of the viral titer from cell lysates. The CGT Viral Vector Lysis Kit with its improved formula gives a consistent, robust, and accurate determination of the final titer. It works in conjunction with the QIAcuity Cell and Gene Therapy dPCR Assays, QIAcuity Digital PCR System, QIAcuity Probe PCR Kit and QIAcuity Nanoplate 8.5K, offering an end-to-end dPCR workflow comparable to qPCR but delivering absolute quantification of AAV vector genome copies in your sample.
Principle
The all-in-one solution for AAV lysis provides:
- Standardization of AAV lysis with much easier standard operating procedure (SOP) implementation and QC
- Consistent quantification of titers
- Broad range of detection between 2.5 copies/µl to 15,000 copies/µl in QIAcuity Nanoplate 8.5k
- Robustness of <10% CV between operators and assays
The CGT Viral Vector Lysis Kit in conjunction with the QIAcuity Cell and Gene Therapy dPCR Assays and the QIAcuity Probe PCR Kit running either singleplex or multiplex reactions on the QIAcuity instruments, now delivers a complete viral titer workflow.
The principle of the dPCR reaction in nanoplates is described here.
Procedure
The CGT Viral Vector Lysis Kit is provided with reagents for 100 or 1000 reactions in two boxes for AAV vectors. The kit is suitable for the lysis of AAV2, AAV5, AAV6, AAV8 and AAV9. The lysates are optimized for QIAcuity Cell and Gene Therapy dPCR Assays in combination with the QIAcuity Probe PCR Kit. These assays enable singleplex as well as multiplex cell and gene therapy applications, including viral titer and vector copy number measurements. The kit does not include restriction enzymes (for e.g., Hpall) that are stated in the protocol (included in the Resources section below).
Applications
The CGT Viral Vector Lysis Kit is used for the lysis of AAV and adenoviruses for a range of applications including viral vector genome titer and vector copy number measurements.
Supporting data and figures
ITR digestion improves the quantification of ITR and non-ITR targets
Two AAV2 samples were processed using the CGT Viral Vector Lysis Kit and quantified using the QIAcuity Digital PCR instrument with 8.5k Nanoplates and the CGT dPCR Assays. The CGT dPCR Assays were run in triplex reactions in the FAM, HEX and Cy5 channels. The samples were serially diluted in 6 steps from 15,000 copies/µL down to 2.5 copies/µL with an R2=1.0 on 8.5k Nanoplates (A, B). Each dilution was measured in technical triplicates. Quantifications were performed with (+) and without (–) restriction digest of the ITR regions. (A) For the titration of a purified AAV2 reference standard sample, the CGT dPCR assays targeting the CMV enhancer bGH polyA regions were used. The expected copies are based on an ITR estimate determined by qPCR measurements from the reference standard supplier and not directly comparable to dPCR measurements. (B) For the titration of the AAV2-SV40 harvest sample, the CGT dPCR Assays targeting the SV40 promoter, SV40 polyA and bGH polyA regions were used.
Resources
Scientific Posters (3)
Kit Handbooks (1)
Brochures & Guides (1)
Application Notes (2)
Quick-Start Protocols (2)
Safety Data Sheets (1)
Certificates of Analysis (1)
FAQ
Do you have to use a restriction enzyme to relieve ITR secondary structures and increase target accessibility?
Do you need to dilute the lysates before setting up the PCR mix?
Do I need to use Proteinase K in the CGT Viral Vector Lysis Kit? What is the recommended concentration?
Is the kit compatible with unpurified in-process samples?
Can I use the lysate from the CGT Viral Vector Lysis kit and combine it with another PCR system?
How do I know if the CGT dPCR assays are compatible with the sample DNA of interest?
Are the CGT dPCR assays also compatible with qPCR?
Is it possible to store the lysates? How can the lysates be stored?
Can I use MspI or HpaII restriction enzymes from other suppliers?
How many AAV genome copies can I load into the QIAcuity?
Can the CGT Viral Vector Lysis kit be used with assays of other suppliers or custom designed assays?
What is the recommended HpaII concentration?
Do you recommend storage of highly diluted AAV samples?