CGT Viral Vector Lysis Kit

用于病毒载体裂解,如不同的腺相关病毒 (AAV) 血清型和腺病毒

Products

CGT Viral Vector Lysis Kit 旨在用于分子生物学应用。该产品不能用于疾病诊断、预防和治疗。
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Cell and Gene Therapy Viral Vector Lysis Kit (100 rxn)

Cat. No. / ID:  250272

该试剂盒包括:CGT 样本稳定剂、DNase I、无核酸酶水、CGT 裂解缓冲液、CGT DNase I 缓冲液和 CGT 稀释缓冲液,足以处理 100 份 AAV 样本 
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Recommended restriction enzyme HpaII is not included in the kit.
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Cell and Gene Therapy Viral Vector Lysis Kit (1000 rxn)

Cat. No. / ID:  250273

该试剂盒包括:CGT 样本稳定剂、DNase I、无核酸酶水、CGT 裂解缓冲液、CGT DNase I 缓冲液和 CGT 稀释缓冲液,足以处理 1000 份 AAV 样本 
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Recommended restriction enzyme HpaII is not included in the kit.

Features

  • 使用一个 AAV 裂解试剂盒和一种方案使当前工作流程的标准化和质量控制 (Quality Control, QC)成为可能
  • 对多种血清型(如 AAV 血清型)的病毒滴度进行一致且可重复的测定
  • 与 QIAcuity Cell and Gene Therapy (CGT) dPCR Assay 相结合的完整工作流程,可在 QIAcuity Digital PCR System 上实现 AAV 滴度的准确测定

Product Details

腺相关病毒是在基因治疗应用领域广泛使用的一种病毒载体。然而,病毒载体的生产和纯化过程需要精密的质量控制,以确保临床研究或患者治疗期间给药的安全性和可靠性。准确定量载体滴度以及确定污染的能力是安全且有效的基于 AAV 的基因治疗的关键。 


随着 CGT Viral Vector Lysis Kit(包含用于 100 或 1000 次反应的试剂)的推出,我们现在拥有了一个经过优化的和标准化的病毒载体裂解工作流程,可以准确且精密地测定病毒滴度。该试剂盒也可用于腺病毒裂解。

Performance

我们提供了从进行 AAV 裂解到在细胞裂解液中定量病毒滴度的完整工作流程。CGT Viral Vector Lysis Kit 及其改进的配方可对最终滴度进行一致、稳健且准确的测定。它与 QIAcuity Cell and Gene Therapy dPCR AssayQIAcuity Digital PCR SystemQIAcuity Probe PCR KitQIAcuity Nanoplate 8.5K 配套使用时,不仅可提供与 qPCR 类似的端到端 dPCR 工作流程,还能够对样本中的 AAV 载体基因组拷贝进行绝对定量。

Principle

AAV 裂解的一体化解决方案提供:

  • AAV 裂解的标准化,具有更容易实施的标准操作程序 (SOP) 和 QC
  • 病毒滴度定量的一致性
  • 检测范围的宽广性,在 QIAcuity Nanoplate 8.5k 中为 2.5 拷贝/µl 至 15,000 拷贝/µl
  • 稳健性,操作员和检测方案之间的变异系数 (CV) <10%

CGT Viral Vector Lysis Kit 与 QIAcuity Cell and Gene Therapy dPCR Assay 以及 QIAcuity Probe PCR Kit 相结合,可在 QIAcuity 仪器上运行单重或多重反应,从而提供完整的病毒滴度工作流程。

有关纳米微孔板中 dPCR 反应的原理说明,请参见这里

 

Procedure

CGT Viral Vector Lysis Kit 在两个盒子中提供了用于 100 或 1000 次 AAV 载体裂解的试剂。该试剂盒适用于 AAV2、AAV5、AAV6、AAV8 和 AAV9 的裂解。裂解物经过优化,可结合 QIAcuity Probe PCR Kit 和 QIAcuity Cell and Gene Therapy dPCR Assay,用于AAV病毒滴度定量。这些检测方案能够实现单重以及多重细胞和基因治疗应用,包括病毒滴度和载体拷贝数测定。该试剂盒不包括方案(参见下面的“资源”部分)中所述的限制酶(例如 Hpall)。

Applications

CGT Viral Vector Lysis Kit 用于 AAV 和腺病毒的裂解以供一系列应用使用,包括病毒载体基因组滴度和载体拷贝数测定。

Supporting data and figures

Resources

试剂盒操作手册 (1)
产品介绍与指南 (1)
快速启动实验方案 (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)
Brochures & Guides (1)
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. 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.
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

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