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Detection method of titer of recombinant lentivirus

A technology of recombinant lentivirus and detection method, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of large sample demand, unsuitable target cells, and poor data repeatability, so as to improve repeatability and Accuracy, effect of reducing sample size

Inactive Publication Date: 2013-01-30
ARMY MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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

Method used

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  • Detection method of titer of recombinant lentivirus
  • Detection method of titer of recombinant lentivirus
  • Detection method of titer of recombinant lentivirus

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Embodiment

[0030] Example Detection method of recombinant lentivirus titer

[0031] 1. Materials used

[0032] 1. Reagents

[0033] DMEM cell culture medium (high glucose) Gibcol;

[0034] Fetal bovine serum Gibcol;

[0035] Human 293FT cells Invitrogen;

[0036] Cell Genomic DNA Extraction Kit Tiangen Company;

[0037] Real-time PCR Master Mix TOYOBO;

[0038] rTaq enzyme Takara;

[0039] HBSS buffer Sigma;

[0040] PBS buffer Gibcol;

[0041] Two recombinant lentiviral suspensions expressing marker gene (eGFP) are prepared by yourself.

[0042] 2. Consumables used

[0043] 24-well cell culture plate Corning;

[0044] Pipette tips of various specifications Axygen;

[0045] Centrifuge tube Axygen;

[0046] 3. Main equipment used

[0047]Flow cytometer detector Beckman Coulter, USA;

[0048] Real-time PCR instrument Bio-rad;

[0049] CO2 incubator Thermo;

[0050] 2. Experimental method

[0051] 1. Determination of control virus titer by FACS

[0052] Sufficiently susp...

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Abstract

The invention discloses a detection method of the titer of a lentivirus carrier, which particularly comprises the following steps: detecting the titer of a control virus by a flow cytometric sorting method, allowing the virus to be detected and the control virus to infect target cells with the same amount under the same condition, extracting total DNA of the target cells infected by the virus to be detected and the control virus respectively, obtaining a ratio R of the relative contents of the genomes of the virus to be detected and the control virus in the genomes of the target cells by relative quantification PCR detection; multiplying the obtained titer of the control virus with the ratio R of the relative contents of the virus to be detected and the control virus to obtain the titer of the virus to be detected which is the titer of the recombinant lentivirus. The invention is applicable to the titer test of a recombinant lentivirus without marker genes, and can accurately determine the titer of the recombinant lentivirus against target cells which has less amounts and can not be largely cultured and propagated in vitro.

Description

technical field [0001] The invention relates to the field of biological genes, in particular to a titer detection method of a lentiviral vector. Background technique [0002] Lentiviral 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. 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. [0003] Existing methods for determining the titer of recombinant lentiviruses include flow cytometry (FACS), enzyme-linked immunoassay (ELISA) and absolute quantitative PCR. [0004] The FACS method is the most accurate method for determining lentivirus titer. In this method, the target cells are first infected with a virus suspension, and then the proportion of successfully infected cells in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70
Inventor 魏泓王勇刘宇郭科男刘昌峨江雯周晓杨刘勤王露露
Owner ARMY MEDICAL UNIV
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