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CGT Viral Vector Lysis Kit

さまざまな血清型のアデノ随伴ウイルス(AAV)およびアデノウイルスなどウイルスベクター溶解物に対応

Products

CGT Viral Vector Lysis Kitは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。
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Cell and Gene Therapy Viral Vector Lysis Kit (100 rxn)

Cat. No. / ID:   250272

本キットの構成品:CGT サンプルスタビライザー、DNase I、ヌクリアーゼフリー水、CGT 溶解バッファー、CGT DNase I バッファー、CGT 希釈用バッファー、100 AAVサンプルを処理可能 
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

本キットの構成品:CGT サンプルスタビライザー、DNase I、ヌクリアーゼフリー水、CGT 溶解バッファー、CGT DNase I バッファー、CGT 希釈用バッファー、1000 AAVサンプルを処理可能 
Recommended restriction enzyme HpaII is not included in the kit.

特徴

  • 1つのキットとプロトコールでAAV溶解物に対応することで、現在のワークフローの標準化と品質管理(QC)が可能
  • 複数の血清型(AAV血清型など)において一貫した再現性のあるウイルス力価測定を実現
  • QIAcuity Cell and Gene Therapy (CGT) dPCR Assaysとの組み合わせによる完全なワークフローと、QIAcuity Digital PCR Systemを使用した正確なAAV力価測定

製品詳細

アデノ随伴ウイルスは、遺伝子治療アプリケーションで広く使用されるウイルスベクターです。しかし、臨床試験や患者の治療において安全かつ信頼できる方法で投与するには、ウイルスベクターの作製および精製プロセスにおける厳密な品質管理が要求されます。ウイルス力価を正確に定量し、コンタミネーションを特定できることが、AAVを活用した安全で有効な遺伝子治療のカギとなります。 


CGT Viral Vector Lysis Kitには100回または1000回分の反応試薬が含まれています。CGT Viral Vector Lysis Kitを取り入れることで、正確で精密なウイルス力価測定が可能になり、ウイルスベクター溶解物のための最適で標準化されたワークフローがもたらされます。本キットはアデノウイルスにも使用できます。

パフォーマンス

AAV溶解から細胞溶解液のウイルス力価定量まで、完全なワークフローを提供します。CGT Viral Vector Lysis Kitは、その優れた組成により、一貫した堅牢で正確な最終力価の測定を可能にします。QIAcuity Cell and Gene Therapy dPCR AssaysQIAcuity Digital PCR SystemQIAcuity Probe PCR Kitおよび QIAcuity Nanoplate 8.5Kと連携して動作し、qPCRと同等のエンドツーエンドdPCRワークフローを提供しながら、サンプル中のAAVベクターゲノムコピーを絶対定量することができます。

原理

次の特長を備えた、AAV溶解のためのオールインワンソリューションです。

  • 標準作業手順書(Standard Operating Procedure、SOP)およびQCを大幅に簡易化してAAV溶解を標準化
  • 一貫したウイルス力価定量
  • QIAcuity Nanoplate 8.5kにおいて、2.5 copies/µlから15,000 copies/µlの幅広い検出範囲を提供
  • オペレータ間およびアッセイ間でCV < 10%の高い堅牢性

CGT Viral Vector Lysis Kitは、QIAcuity Cell and Gene Therapy dPCR AssaysおよびQIAcuity Probe PCR Kitと組み合わせてシングルプレックスまたはマルチプレックス反応に使用することで、完全なウイルス力価測定ワークフローが実現します。

ナノプレートでのdPCRの原理はこちらで説明しています。

 

操作手順

CGT Viral Vector Lysis Kitは、AAVベクター用の反応試薬100回分または1000回分を2つの箱で提供します。本キットは、AAV2、AAV5、AAV6、AAV8およびAAV9の溶解物に適しています。溶解液は、QIAcuity Cell and Gene Therapy dPCR AssaysとQIAcuity Probe PCR Kitを組み合わせた分析に最適です。これらのアッセイを使用することで、ウイルス力価測定やウイルスベクターコピー数測定など、細胞遺伝子治療アプリケーションにおけるシングルプレックスおよびマルチプレックス分析が可能です。本キットには、プロトコール(下記のResourcesを参照)に記載の制限酵素(Hpallなど)は含まれません。

アプリケーション

CGT Viral Vector Lysis Kitは、AAVおよびアデノウイルスの溶解物に使用され、ウイルスベクターのゲノム力価測定やコピー数測定など幅広いアプリケーションに活用されます。

裏付けデータと数値

リソース

キットハンドブック (1)
パンフレット (1)
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.
クイックスタートプロトコール (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
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