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Method for detecting combination of lentivirus quality indicators and its application

A quality index, lentivirus technology, applied in the field of titer detection of lentiviral vectors, can solve problems such as poor data repeatability, large sample demand, unsuitable target cells, etc., and achieve low cost, good specificity, and high sensitivity Effect

Active Publication Date: 2021-09-28
NANJING IASO BIOTHERAPEUTICS CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires a large amount of samples, many influencing factors, and poor data reproducibility. It is not suitable for target cells that cannot be cultured in large quantities in vitro.

Method used

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  • Method for detecting combination of lentivirus quality indicators and its application
  • Method for detecting combination of lentivirus quality indicators and its application
  • Method for detecting combination of lentivirus quality indicators and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: The method for detecting the total copy number of virus RNA by real-time fluorescent quantitative qPCR

[0057] The qPCR primers for detecting the total copy number of lentiviral RNA are SEQ ID No1-No 17, wherein the primers of SEQ ID No1-No9 are specific to the EF-1a promoter located in the vector plasmid, and the primers of SEQ ID No 10-No 13 are specific to the EF-1a promoter located in the vector plasmid The primers of WPRE, SEQ ID No14-17 are specific to the CD19-specific chimeric antigen receptor (CAR) part.

[0058] Specific steps are as follows:

[0059] (1) Extraction of template DNA: the total DNA of the lentivirus sample to be tested was extracted with a viral RNA / DNA extraction kit (MiniBEST Viral RNADNA Extraction Kit from Takara). See the kit instructions for specific steps. DNA was diluted to 200ng / μL and 100ng / μL. (2) Reverse transcription reaction (RT): Using a reverse transcription kit (Takara PrimeScript RTMaster Mix (Perfect Real Tim...

Embodiment 2

[0077] Example 2: Method for detecting the copy number of virus integrated into chimeric antigen receptor T cells by real-time fluorescent quantitative qPCR

[0078] The qPCR primers for detecting the copy number of virus integrated into T cells are SEQ ID No18-No 23. The templates of the primers of SEQ ID No18-No21 are T cell genomes, which are determined by qPCR; the templates corresponding to the primers of SEQ ID No22-23 are T cell RNAs; RT-qPCR is to be performed.

[0079] Specific steps are as follows:

[0080] (1) Extraction of template DNA or RNA: Extract the DNA or RNA of the chimeric antigen receptor T cell sample to be tested with a viral RNA / DNA extraction kit (MiniBEST ViralRNA DNA Extraction Kit from Takara), and dilute each DNA or RNA sample to 200ng / μL and 100ng / μL.

[0081] (2) Gradient dilution of the standard: Calculate the initial standard copy number / ul of the vector plasmid according to the following formula:

[0082]

[0083] The standard for detec...

Embodiment 3

[0094] Example 3: Method for detecting the copy number of replication-competent lentivirus by real-time fluorescent quantitative qPCR

[0095] The qPCR primers for detecting the copy number of the replication-competent lentivirus are SEQ ID No24-No 27, and the primers are located at VSV-G of the envelope plasmid.

[0096] Specific steps are as follows:

[0097] (1) Extraction of template DNA: DNA of the sample to be tested was extracted with a viral RNA / DNA extraction kit (Takara's MiniBEST Viral RNADNA Extraction Kit). DNA was diluted to 200ng / ul and 100ng / ul.

[0098] (2) Gradient dilution of the standard: Calculate the copy number of the initial standard of the vector plasmid / μL according to the following formula

[0099]

[0100] Prepare a series of dilution standards with different copy numbers by serial dilution, each copy number is 10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1

[0101] The standard for measuring the copy number of replication-competent lentiviru...

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Abstract

The invention belongs to the field of biotechnology, in particular to a titer detection method of a lentiviral vector. Disclosed are a quality index detection method for real-time fluorescent quantitative nucleic acid amplification detection (qPCR and RT-qPCR) using sequence-specific primers and fluorescent dyes, primers used, and standard items. The determination method is efficient, accurate, easy to operate, small in sample consumption and good in repeatability, and solves the limitation problem of inaccurate vector quantification in the field of eukaryotic genetic engineering and gene therapy. It has short detection time, simple operation steps, and no post-amplification analysis process. Compared with the Taqman probe method, SYBR Green I does not need to design and synthesize fluorescent probes, which simplifies design and reduces costs. Combined with melting curve analysis , to judge the specificity of the reaction.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a titer detection method of a lentiviral vector. Background technique [0002] Lentiviral vectors are a type of retroviral vectors. Compared with traditional viral vectors, lentiviral vectors are evenly distributed in the genome, thereby reducing the chance of activating the host's endogenous genes. Viral vectors are currently one of the most widely used gene delivery tools, and have shown broad application prospects in gene therapy research and the preparation of transgenic animals. [0003] In the prior art, the main methods for measuring lentivirus titer include flow cytometry (FACS), enzyme-linked immunoassay (ELISA) method, gold standard test paper method and quantitative PCR method. Among them, the FACS method is that this method can accurately measure the actual infectivity of the recombinant lentivirus, and the measured titer data can accurately reflect the actual value of t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/702C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/114C12Q2537/16
Inventor 杨永坤孟广荣
Owner NANJING IASO BIOTHERAPEUTICS CO LTD