Method for detecting slow virus quality index combination and application of method

A quality index and lentivirus technology, which is applied in the titer detection field of lentiviral vectors, can solve the problems of many influencing factors, poor data repeatability, large sample demand, etc., and achieve the effect of high sensitivity

Active Publication Date: 2018-06-01
NANJING IASO BIOTHERAPEUTICS CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 slow virus quality index combination and application of method
  • Method for detecting slow virus quality index combination and application of method
  • Method for detecting slow virus quality index combination and application of method

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, particularly relates to a titer detection method for a slow virus carrier, and discloses a quality index detecting method conducting real-time fluorescence quantification nucleic acid amplification detection (qPCR and RT-qPCR) by using a sequence specific primer and a fluorescent dye, a used primer and a standard product. The detecting method isefficient and accurate, the operation is easy, the use amount of a sample is low, the repeatability is good, and the limit problem of inaccurate carrier quantitation in the fields of eucaryon geneticengineering and gene therapy is solved. The method has the advantages of detection time and simple and convenient operation step, the analysis process after the amplification is not needed, comparedwith the Taqman probe method, the SYBR Green I does not need the design of a synthesis fluorescent probe, the design is simplified, the cost is lowered, and a dissolution curve is combined in the 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 Applications(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
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