CN111334565A

CN111334565A - Method for detecting CAR gene copy number in T lymphocyte by fluorescent quantitative PCR

Info

Publication number: CN111334565A
Application number: CN202010242228.7A
Authority: CN
Inventors: 樊克兴; 危华峰; 卫静
Original assignee: Shili Biotechnology Co ltd
Current assignee: Shili Biotechnology Co ltd
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2020-06-26

Abstract

The invention discloses a method for detecting the copy number of a CAR gene in a T lymphocyte by fluorescent quantitative PCR, which comprises the following specific steps: s1: preparing a standard substance, namely using a plasmid containing the CAR gene as the standard substance, and performing gradient dilution on the standard substance by 10 times; s2: extracting DNA of a sample to be detected; s3: carrying out real-time fluorescent quantitative PCR reaction on the DNA of the standard substance series and the sample to be detected by using a primer pair and a probe; s4: and substituting the Ct value of the CAR gene of the sample to be detected into a calculation formula of a standard curve in S3, and standardizing the content of the correction gene by taking the internal reference gene as a reference. The primer pair, the probe or the method disclosed by the invention have the advantages of good specificity, high sensitivity, good repeatability of a reaction system and good stability, and can be used for quantitative detection of the copy number of CAR in the prepared CAR-T or quantitative detection of the copy number of CAR in peripheral blood cells of a CAR-T treatment patient.

Description

Method for detecting CAR gene copy number in T lymphocyte by fluorescent quantitative PCR

Technical Field

The invention relates to the technical field of CAR gene copy number detection, in particular to a method for detecting CAR gene copy number in T lymphocytes by fluorescence quantitative PCR.

Background

With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) (June CH, Blazar BR, Riley JL. engineering lymphocytic subsets: tools, tris and tribulions. NatRevImmunol.2009; 9:704-16) became one of the most promising tumor immunotherapies. CAR-T cell therapy expresses a fusion protein of Single chain antibody (scFv) for recognizing tumor-associated antigen and T cell activation sequence on the surface of T cell by exogenous gene transfection technology, so that the scFv capable of specifically recognizing tumor-associated antigen is coupled with activation proliferation signal domain in T cell via transmembrane region. CAR-expressing T cells bind tumor antigens in an antigen-dependent, but not MHC-restricted manner, initiating and activating a specific killing tumor response.

Construction of CAR-T cells requires transfection and integration of the CAR gene into the T cell genome by either viral or non-viral systems. When the gene is normally expressed, a transmembrane CAR structure is formed on the cell membrane, and CAR-T cells have the activity of recognizing and killing target cells. Therefore, accurate detection of the copy number of the CAR gene is a critical step in CAR-T drug quality control and an important link in diagnosis. The traditional method for detecting the copy number of the exogenous gene is Southern blot, but is time-consuming, labor-consuming and requires a large amount of genomic DNA. The real-time fluorescent quantitative PCR technology is a DNA/RNA quantitative technology which is rapidly developed in recent years, omits the complicated steps of a southern blot method, and can simply, rapidly and accurately detect the copy number of a genome.

The TaqMan quantitative PCR method is based on the original pair of primers, a specific probe combined with a target sequence between forward and reverse primers needs to be synthesized again, a fluorescent group is connected to the 5 'end of the probe, and a quenching group is arranged at the 3' end. When the probe is matched with the target sequence, the fluorescence emitted by the fluorescent group is quenched due to the proximity of the quenching group at the 3' end; during PCR amplification, the 5 '-3' exonuclease activity of Taq enzyme cuts and degrades the probe, so that the report fluorescent group and the quenching fluorescent group are separated, a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescent molecule is formed when one DNA chain is amplified, and the accumulation of the fluorescence signal and the formation of a PCR product are completely synchronous.

Disclosure of Invention

The invention aims to provide a method for detecting the copy number of a CAR gene in a T lymphocyte by fluorescence quantitative PCR, which comprises the following specific steps:

s1: preparing a standard substance, namely using a plasmid containing CAR gene as the standard substance, performing gradient dilution on the plasmid by 10 times, calculating the copy number of the plasmid, extracting Uninfected (UNT) T lymphocyte DNA as a background DNA template, performing gradient dilution to prepare a standard substance series, and preparing a standard curve;

s2: extracting DNA of a sample to be detected;

s3: using a primer pair and a probe to carry out real-time fluorescence quantitative PCR reaction on the DNA of the standard substance series and the sample to be detected, wherein the conditions of the PCR amplification reaction are as follows: 95 deg.C for 10 min; at 95 deg.C, 15s, 60 deg.C for 1min for 40 cycles; drawing a standard curve according to the Ct value of the RSV gene generated by each standard substance and the corresponding copy number logarithm value to obtain a calculation formula of the standard curve;

s4: and (3) substituting the Ct value of the CAR gene of the sample to be detected into a calculation formula of a standard curve in S3, standardizing and correcting the content of the gene by taking the internal reference gene as a reference, avoiding inaccurate results caused by sample loading amount and experimental errors in the sample loading process, and obtaining the absolute copy number of the CAR gene of the sample to be detected.

Preferably, the PCR reaction in S3 is performed using a kit, and the kit comprises: primer pair 1 and primer pair 2, and probe a and probe B; the kit also comprises reagents required by PCR reaction, specifically Mg2+, reaction buffer, dNTP and Taq enzyme.

Preferably, the primer 1 and the probe A are designed by selecting RSV promoter specific sequences on a vector containing CAR genes, and the primer pair 2 and the probe B are designed by selecting PTBP2 genes as reference genes.

Preferably, the 5 'end of the probe A and the 3' end of the probe B are marked with a fluorescence reporter group, and the fluorescence reporter group is selected from FAM and VIC; the fluorescence quenching group is selected from TAMRA.

Compared with the prior art, the invention has the beneficial effects that: the primer pair, the probe or the method has universality on CAR gene detection, the primer is designed in an RSV promoter region, and the method is suitable for copy number identification of any CAR gene containing an RSV promoter viral vector and has no limitation on CAR structure. The primer pair, the probe or the method disclosed by the invention have the advantages of good specificity, high sensitivity, good repeatability of a reaction system and good stability, and can be used for quantitative detection of the copy number of CAR in the prepared CAR-T or quantitative detection of the copy number of CAR in peripheral blood cells of a CAR-T treatment patient. The primer pair, probe or method of the invention detects CAR at a minimum effective detection concentration of 0.328 copies/cell.

Drawings

FIG. 1 is a graph of Uninfected (UNT) T lymphocyte DNA prepared as a standard;

FIG. 2 is a graph of Ct value of RSV gene plotted against the corresponding log copy number standard;

FIG. 3 is a viral vector plasmid map of the CAR gene;

FIG. 4 is a graph showing the cycle number of the fluorescent quantitative PCR amplification reaction.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1

A kit, and the kit comprises: primer pair 1 and primer pair 2, and probe a and probe B; the kit also comprises reagents required by PCR reaction, specifically Mg2+, reaction buffer, dNTP and Taq enzyme. Primer 1 and probe A are designed by selecting RSV promoter specific sequences on a vector containing CAR gene, and primer pair 2 and probe B are designed by selecting PTBP2 gene as reference gene. The 5 'ends of the probes A and B are marked with fluorescence reporter groups, the 3' ends of the probes A and B are marked with fluorescence quenching groups, and the fluorescence reporter groups are selected from FAM and VIC; the fluorescence quenching group is selected from TAMRA. The primers and sequence information are as follows:

example 2

A method for detecting the copy number of a CAR gene in a T lymphocyte by fluorescence quantitative PCR comprises the following specific steps:

s2: extracting DNA of a sample to be detected;

The primer pair, the probe or the method have universality on the detection of the CAR gene, the primer is designed in an RSVpromoter region, the primer is suitable for the copy number identification of any CAR gene containing an RSV promoter virus vector, and the CAR structure is not limited. The primer pair, the probe or the method disclosed by the invention have the advantages of good specificity, high sensitivity, good repeatability of a reaction system and good stability, and can be used for quantitative detection of the copy number of CAR in the prepared CAR-T or quantitative detection of the copy number of CAR in peripheral blood cells of a CAR-T treatment patient. The primer pair, probe or method of the invention detects CAR at a minimum effective detection concentration of 0.328 copies/cell.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims

1. A method for detecting the copy number of CAR gene in T lymphocyte by fluorescent quantitative PCR is characterized in that: the method for detecting the copy number of the CAR gene in the T lymphocyte by fluorescent quantitative PCR comprises the following specific steps:

s2: extracting DNA of a sample to be detected;

2. The method for detecting the copy number of the CAR gene in the T lymphocyte through the fluorescent quantitative PCR according to claim 1, wherein the method comprises the following steps: the PCR reaction in S3 is performed by using a kit, and the kit comprises: primer pair 1 and primer pair 2, and probe a and probe B; the kit also comprises reagents required by PCR reaction, specifically Mg2+, reaction buffer, dNTP and Taq enzyme.

3. The method for detecting the copy number of the CAR gene in the T lymphocyte through the fluorescent quantitative PCR according to claim 1, wherein the method comprises the following steps: primer 1 and probe A are designed by selecting RSV promoter specific sequences on a vector containing CAR gene, and primer pair 2 and probe B are designed by selecting PTBP2 gene as reference gene.

4. The method for detecting the copy number of the CAR gene in the T lymphocyte through the fluorescent quantitative PCR according to claim 1, wherein the method comprises the following steps: the 5 'ends of the probes A and B are marked with fluorescence reporter groups, the 3' ends of the probes A and B are marked with fluorescence quenching groups, and the fluorescence reporter groups are selected from FAM and VIC; the fluorescence quenching group is selected from TAMRA.