Viral Vector Direct Lysis Kits

For the direct lysis of viral vectors, such as different adeno-associated virus (AAV) serotypes and adenoviruses and lentivirus (LV)

S_1084_5_GEN_V2

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Cell and Gene Therapy Lentivirus Lysis Kit (100 rxn)

Cat no. / ID.   250323

For the lysis of lentivirus (LV). The kit includes: DNase I, RDD buffer, Proteinase K, CGT Dilution Buffer, Nuclease-free Water, CGT PBS buffer, enough to process 100 LV samples
Kit
LV lysis
AAV lysis
Size
100 reactions
1000 reactions
The Cell and Gene Therapy Lentivirus Lysis Kit (100 rxn) is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
The Viral Vector Direct Lysis Kits is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • One kit and one protocol solution for AAV or LV lysis enables standardization and quality control (QC) of current workflows
  • Consistent and reproducible measurement of viral titers for multiple serotypes (e.g., AAV serotypes) or of different purities (e.g., from in-process supernatant to highly purified samples)
  • Complete workflow combined with QIAcuity Cell and Gene Therapy (CGT) dPCR Assays and accurate measurement of AAV or LV titers on the QIAcuity Digital PCR System

Product Details

Adeno-associated virus and lentivirus are widely used viral vectors in cell and 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 and LV-based therapies.  

With the introduction of direct vector lysis solutions for AAV or LV that include reagents for either 100 or 1000 reactions, we now have an optimized and standardized workflow for direct viral vector lysis through to an accurate and precise way of determining the viral titer.

 

Performance

We offer a complete workflow from AAV or LV particle lysis to quantification of the viral titer. The CGT Viral Vector Lysis Kit and the CGT Lentivirus Lysis Kit, with their unique formulas, give a consistent, robust, and accurate determination of the vector titer while saving hands-on time in comparison to the current workflow with a separate sample preparation step. A standardized sample processing step is key to reliable genome titration.  

It works in conjunction with the QIAcuity Cell and Gene Therapy dPCR Assays, QIAcuity Digital PCR System, QIAcuity Probe PCR Kit (cat. nos. 250272, 250273), QIAcuity OneStep Advanced Probe Kit (cat. nos. 250323, 250324), and QIAcuity Nanoplate 8.5K, offering an end-to-end dPCR workflow comparable to qPCR but delivering absolute quantification of vector genome copies in your sample. 

 

Principle

The all-in-one solution for viral vector lysis provides:

  • Standardization of vector lysis with much easier standard operating procedure (SOP) implementation and QC
  • Consistent quantification of titers independent of sample purities or serotypes
  • Broad range of detection down to 2.5 copies/µL in QIAcuity Nanoplate 8.5k

The CGT viral vector lysis kits, in conjunction with the QIAcuity Cell and Gene Therapy dPCR Assays and dedicated master mixes running either singleplex or multiplex reactions on the QIAcuity instruments, now deliver a complete viral titer workflow.

The principle of the dPCR reaction in nanoplates is described here. 

 

Procedure

The CGT Viral Vector Lysis Kit (cat. nos. 250272, 250273) 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 (cat. nos. 250272, 250273).

The CGT Lentivirus Lysis Kit (cat. nos. 250323, 250324) is provided with reagents for 100 or 1000 reactions in one box for LV vectors. The lysates are optimized for QIAcuity Cell and Gene Therapy dPCR Assays in combination with the QIAcuity OneStep Advanced Probe Kit (cat. nos. 250323, 250324).

These assays enable singleplex as well as multiplex cell and gene therapy applications. The kit does not include restriction enzymes (e.g., Hpall) stated in the protocol (included in the Resources section below). 

 

Applications

The CGT Viral Vector Lysis Kit (cat. nos. 250272, 250273) is used for the lysis of AAV and adenoviruses. The CGT Lentivirus Lysis Kit (cat. nos. 250323, 250324) is used for the lysis of LV. These kits are optimized for a range of applications, including viral vector genome titer.

 

Supporting data and figures

Resources

Application Notes (2)
The QIAcuity dPCR System, complemented by the QIAcuity Software Suite version 2.5 and higher, offers a powerful solution for determining DNA integrity and stability. Analyzing up to 5 targets simultaneously enables accurate and precise integrity assessment. The importance of determining genome integrity becomes particularly evident in analyzing viral vectors such as AAVs, which are known for their susceptibility to packaging errors. Moreover, dPCR is a valuable tool for assessing DNA stability, providing valuable insights into storage and processing impacts.
The QIAcuity dPCR System, complemented by the QIAcuity Software Suite version 2.5 and higher, offers a powerful solution for determining DNA integrity and stability. Analyzing up to 5 targets simultaneously enables accurate and precise integrity assessment. The importance of determining genome integrity becomes particularly evident in analyzing viral vectors such as AAVs, which are known for their susceptibility to packaging errors. Moreover, dPCR is a valuable tool for assessing DNA stability, providing valuable insights into storage and processing impacts.
Quick-Start Protocols (2)
This protocol is optimized for the processing of viral vectors for genome titer quantification on the QIAcuity® digital PCR instrument using the dPCR CGT Viral Vector Lysis Kits (cat. no. 250272, 250273). The kits are optimized for usage with the QIAcuity Probe PCR Kit (cat. no. 250101, 250102, 250103) and the QIAcuity Cell and Gene Therapy (CGT) dPCR Assays
This protocol is optimized for the processing of viral vectors for genome titer quantification on the QIAcuity® digital PCR instrument using the dPCR CGT Viral Vector Lysis Kits (cat. nos. 250272, 250273). The kits are optimized for usage with the QIAcuity Probe PCR Kit (cat. nos. 250101, 250102, 250103) and the QIAcuity Cell and Gene Therapy (CGT) dPCR Assays.
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?
It is highly recommended and an important step in the protocol. ITR secondary structures reduce target accessibility and negatively affect quantification of ITR targets and non-ITR targets.
We recommend using the following HpaII restriction enzymes: Thermo Scientific™ HpaII (10 U/µL, cat. nos. ER0511, ER0512) or Invitrogen™ Anza™ 93 HpaII (cat. no. IVGN0936).
FAQ ID - 3974
Do you need to dilute the lysates before setting up the PCR mix?
The lysates must be diluted in total of at least 1:100 taking together the dilution after capsid lysis and the dilution in the PCR reaction. A total of 200x dilution is recommended.
FAQ ID - 3978

Do I need to use Proteinase K in the CGT Viral Vector Lysis Kit? What is the recommended concentration?
No, Proteinase K use is optional and does not interfere with the kit performance. It is recommended to use 2 µL of Proteinase K (RP107B) in a 50 µL reaction. The Proteinase K has a concentration of 20mg/mL with 800 U/mL.
FAQ ID - 3971
Is the kit compatible with unpurified in-process samples?
Yes, it is.
FAQ ID - 3965
Can I use the lysate from the CGT Viral Vector Lysis kit and combine it with another PCR system?
Yes, however, the complete workflow has been optimized with the QIAcuity Cell and Gene Therapy (CGT) dPCR Assays on the QIAcuity Digital PCR System to ensure standardization and reproducibility of viral vector titer determinations.
FAQ ID - 3979

How do I know if the CGT dPCR assays are compatible with the sample DNA of interest?
An extended sequence context for each assay is provided in the corresponding data sheets. 
FAQ ID - 3980

Are the CGT dPCR assays also compatible with qPCR?
Yes, the assays can also be used for vector genome titration in a qPCR setup.
FAQ ID - 3981

Is it possible to store the lysates? How can the lysates be stored?
Long-term storage of lysates is not recommended. If lysates must be stored, it is recommended to store them at 4°C. Do not freeze.
FAQ ID - 3973
Can I use MspI or HpaII restriction enzymes from other suppliers?
The HpaII enzymes from Thermo Fisher Scientific ER0511, ER0512 and IVGN0936 have been extensively tested and show high compatibility with the QIAcuity Probe PCR chemistry. Use of MspI enzymes is not recommended. Compatibility of alternative enzymes must be individually tested and may need additional optimization.
FAQ ID - 3975
How many AAV genome copies can I load into the QIAcuity?
Recommended detection range in the dPCR using a 8.5k nanoplate is between 2.5 cop/μL to 15,000 copies/μL.
FAQ ID - 3977
Can the CGT Viral Vector Lysis kit be used with assays of other suppliers or custom designed assays?
Yes. Optimization of the cycling and imaging conditions might be needed.
FAQ ID - 3982

What is the recommended HpaII concentration?
Enzyme concentration depends on sample input and incubation time. When following our protocol described in the handbook HpaII enzymes work in the following range:
  • IVGN0936: 0.025 – 0.125 U/µL
  • ER0511/ER0512: 0.25 – 0.5 U/µL
FAQ ID - 3976
Do you recommend storage of highly diluted AAV samples?
No, it is not recommended to store AAV samples in a highly diluted state.
FAQ ID - 3972