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Fluorescent probe, primer pair, fluorescent quantitative PCR kit and detection method

A fluorescent probe and fluorescent quantitative technology, applied in the field of medical biology, can solve the problems of strict operating environment requirements, low sensitivity, complex time-consuming repeatability, etc., and achieve the effect of high infection efficiency

Pending Publication Date: 2020-11-10
SHENZHEN BINDEBIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These detection methods generally have the disadvantages of low detection specificity, low sensitivity, complex time-consuming and poor repeatability, and have strict requirements on the operating environment.

Method used

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  • Fluorescent probe, primer pair, fluorescent quantitative PCR kit and detection method
  • Fluorescent probe, primer pair, fluorescent quantitative PCR kit and detection method
  • Fluorescent probe, primer pair, fluorescent quantitative PCR kit and detection method

Examples

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Embodiment 1

[0072] A preparation method of chimeric antigen receptor T cells (CAR-T), comprising the following steps:

[0073] (1) Construction of pWPXLD-CAR recombinant plasmid

[0074] The chimeric antigen receptor (CAR) gene fragment was inserted between the BamH1 and EcoR1 restriction sites downstream of the eukaryotic promoter in the pWPXLD plasmid vector, and the WPRE element was located downstream of the CAR gene fragment. Then it was transferred into Escherichia coli competent cell DH5α, and positive clones were identified by PCR and sequenced. After gel electrophoresis detection and sequencing of PCR products, the size and sequence of the target fragments were identified, and the successful construction of figure 1 The indicated pWPXLD-CAR recombinant plasmid containing the WPRE element and the CAR target gene.

[0075] (2) Construction of recombinant lentivirus

[0076] The pWPXLD-CAR recombinant plasmid, packaging plasmid psPAX2, and envelope plasmid pMD2G were co-transfecte...

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Abstract

The invention provides a fluorescent probe. The fluorescent probe comprises a single-stranded nucleotide fragment, a fluorescent reporter group is marked at the 5'end of the single-stranded nucleotidefragment, a fluorescent quenching group is marked at the 3 'end of the nucleotide fragment, and the single-stranded nucleotide fragment comprises a nucleotide sequence shown as SEQ ID NO: 1. The fluorescent probe has the advantages of high specificity and high sensitivity, and can be used for quickly and quantitatively analyzing the copy number of lentiviruses containing WPRE elements, particularly the copy number of lentiviruses in CAR-T mediated by a lentivirus gene containing the WPRE elements. The invention also provides a primer pair, a fluorescent quantitative PCR kit and a detection method.

Description

technical field [0001] The invention relates to the field of medical biology, in particular to a fluorescent probe, a primer pair, a fluorescent quantitative PCR kit and a detection method. Background technique [0002] Immune cell therapy is the only method that can completely eliminate cancer cells in the existing technology, and is considered to be the most promising treatment method in the comprehensive tumor treatment model in the 21st century. Compared with traditional treatment methods, immune cell therapy technology has the great advantages of strong specificity and almost no toxic side effects in the treatment of malignant tumors. It makes up for the disadvantages of traditional surgery, radiotherapy and chemotherapy, and becomes the fourth tumor treatment method means. Among them, Chimeric Antigen Receptor–Modified T Cells (CAR-T) technology, as one of the latest immune cell therapy technologies, has received extensive attention and research. [0003] At present,...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6851
CPCC12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/114C12N15/11C12Q1/686C12Q1/70
Inventor 唐超张宏玲李浩莎任异菲
Owner SHENZHEN BINDEBIOTECH CO LTD