CN112481361A - Primer set, fluorescent probe set, kit and method for detecting copy number of CAR gene in average single CAR-T cell - Google Patents

Primer set, fluorescent probe set, kit and method for detecting copy number of CAR gene in average single CAR-T cell Download PDF

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CN112481361A
CN112481361A CN202011370271.8A CN202011370271A CN112481361A CN 112481361 A CN112481361 A CN 112481361A CN 202011370271 A CN202011370271 A CN 202011370271A CN 112481361 A CN112481361 A CN 112481361A
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gene
car
detecting
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primer
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焦顺昌
张嵘
梁皓
王雪薇
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Beijing Dingcheng Taiyuan Biotechnology Co ltd
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Abstract

The invention provides a primer group, a fluorescent probe group, a kit and a method for detecting the copy number of a CAR gene in an average single CAR-T cell, belonging to the technical field of biology, wherein the primer group comprises a primer pair for detecting the CAR gene and a primer pair for detecting an internal reference gene; the fluorescent probe set comprises a fluorescent probe for detecting CAR gene and a fluorescent probe for detecting internal reference gene; the kit comprises the primer group and the fluorescent probe group; the primer has a single amplification peak and good specificity, and the accurate range of an amplification curve obtained by using the primer group, the fluorescent probe group and the detection method is 5-5 multiplied by 106Between copies, the detection range of the CAR gene copy number in a single CAR-T cell can reach 10 theoretically on average‑6～106The composition is effective in relieving fruit diseasesThe detection range is wide; has good application prospect.

Description

Primer set, fluorescent probe set, kit and method for detecting copy number of CAR gene in average single CAR-T cell
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer group, a fluorescent probe group, a kit and a method for detecting the copy number of a CAR gene in an average single CAR-T cell.
Background
The CAR-T cell is obtained by the following genetic engineering means: a fusion protein of a Single chain variable (scFv) of an antibody recognizing a tumor-associated antigen and a T cell activation sequence is expressed on the surface of a T cell, and the scFv capable of specifically recognizing the tumor-associated antigen is coupled with an intracellular activation proliferation signal domain of the T cell through a 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.
The preparation and quality control process of the CAR-T cells for clinical treatment are crucial to the curative effect of the CAR-T cells. Sensitive and accurate CAR gene copy number detection methods are essential for CAR-T cell release and clinical monitoring of CAR-T cells. The method for detecting the copy number of the gene by double fluorescence quantitative PCR is characterized in that in a DNA amplification reaction system, amplification products of two genes respectively have two different fluorescent groups, and the copy number of a target gene is calculated by performing accumulation statistics on two fluorescent signals and simultaneously monitoring the product expression quantity of the two genes in the system in real time. Compared with the single-gene detection system which is applied more at present, the dual-fluorescence quantitative PCR has the advantages of high sensitivity, simple operation and the like.
The detection of the copy number of the CAR gene in an average single CAR-T cell by using double fluorescence quantitative PCR is a better choice, but the current method has the problems of poor specificity and narrow detection range.
Disclosure of Invention
In view of the above, the present invention aims to provide a primer set, a fluorescent probe set, a kit and a method for detecting the copy number of a CAR gene in an average single CAR-T cell; the primer has a single amplification peak and good specificity, and the accurate range of an amplification curve obtained by using the primer group, the fluorescent probe group and the detection method is 5-5 multiplied by 106Between copies, the detection range of the CAR gene copy number in a single CAR-T cell can reach 10 theoretically on average-6～106The result is accurate, and the detection range is wide.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a primer group for detecting the copy number of a CAR gene in an average single CAR-T cell, which comprises a primer pair for detecting the CAR gene and a primer pair for detecting an internal reference gene;
the CAR gene comprises a CD3zeta gene and/or a WPRE gene, and the reference gene comprises an ABL gene and/or an IFNG gene;
the nucleotide sequences of the primer pair for detecting the CD3zeta gene are shown as SEQ ID No.1 and SEQ ID No. 2;
the nucleotide sequence of the primer pair for detecting the WPRE gene is shown as SEQ ID No.3 and SEQ ID No. 4;
the nucleotide sequence of the primer pair for detecting the ABL gene is shown as SEQ ID No.5 and SEQ ID No. 6;
the nucleotide sequence of the primer pair for detecting the IFNG gene is shown as SEQ ID No.7 and SEQ ID No. 8.
The invention provides a fluorescent probe set for detecting the copy number of a CAR gene in an average single CAR-T cell, which comprises a fluorescent probe for detecting the CAR gene and a fluorescent probe for detecting an internal reference gene; the CAR gene comprises a CD3zeta gene and/or a WPRE gene, and the reference gene comprises an ABL gene and/or an IFNG gene;
the nucleotide sequence of the fluorescent probe for detecting the CD3zeta gene is shown as SEQ ID No. 9;
the nucleotide sequence of the fluorescent probe for detecting the WPRE gene is shown as SEQ ID No. 10;
the nucleotide sequence of the fluorescent probe for detecting the ABL gene is shown as SEQ ID No. 11;
the nucleotide sequence of the fluorescent probe for detecting the IFNG gene is shown as SEQ ID No. 12.
The invention provides a kit for detecting the copy number of a CAR gene in an average single CAR-T cell, which comprises the primer group and the fluorescent probe group.
Preferably, the primer set for detecting the CD3zeta gene, the primer set for detecting the IFNG gene, the fluorescent probe for detecting the CD3zeta gene and the fluorescent probe for detecting the IFNG gene are included.
Preferably, the kit also comprises a double fluorescent quantitative PCR reaction reagent.
The invention provides a method of detecting the copy number of a CAR gene in an average single CAR-T cell, comprising the steps of:
1) constructing the CAR gene and the reference gene on a PZXK plasmid vector together to obtain a standard plasmid; setting standard plasmids with different concentrations;
2) extracting the whole genome DNA of a sample to be detected, and adjusting the whole genome DNA to a proper concentration range of 1 ng/mul-500 ng/mul;
3) mixing the DNA to be detected, the primer group of the CAR gene, the primer group of the internal reference gene, the fluorescent probe of the CAR gene, the fluorescent probe of the internal reference gene and the dual fluorescent quantitative PCR reaction reagent in the step 2) to obtain a dual fluorescent quantitative PCR reaction system of a sample to be detected;
respectively mixing standard plasmids with different concentrations with a primer group of a CAR gene, a primer group of an internal reference gene, a fluorescent probe of the CAR gene, a fluorescent probe of the internal reference gene and a dual fluorescent quantitative PCR reaction reagent to obtain standard plasmid dual fluorescent quantitative PCR reaction systems with different concentrations;
performing fluorescent quantitative PCR detection on the sample dual-fluorescent quantitative PCR reaction system to be detected and the standard plasmid dual-fluorescent quantitative PCR reaction system with different concentrations on a computer to obtain the Ct value of the CAR gene of the sample to be detected, the Ct value of the reference gene of the sample to be detected, the Ct value of the CAR gene of the standard plasmid with different concentrations and the Ct value of the reference gene of the standard plasmid with different concentrations;
calculating the copy number of the CAR gene in the CAR-T cells of the sample to be detected;
CAR gene copy number in CAR-T cells of samples to be tested is 2-△△Ct×2；
Wherein the delta Ct is equal to the delta Ct-correction coefficient of the sample to be detected;
the delta Ct of the sample to be detected is the Ct value of the CAR gene of the sample to be detected-the Ct value of the reference gene of the sample to be detected;
the correction coefficient is (delta Ct1 +. DELTA Ct2 … …. delta. Ctn)/n;
where Δ Ctn is the Ctn value of the CAR gene of the standard plasmid-the Ctn value of the reference gene in the standard plasmid, and n is a specific concentration.
Preferably, the concentration of the standard plasmids with different concentrations is 1.25 copies/mu l to 1.25 multiplied by 106copies/μl。
Preferably, the double fluorescent quantitative PCR reaction system of the sample to be detected is calculated by 20 mul and comprises the following components:
Figure BDA0002805893000000041
the standard plasmid dual-fluorescence quantitative PCR reaction system is calculated by 20 mu l and comprises the following components:
Figure BDA0002805893000000042
preferably, the reaction procedures of the double fluorescent quantitative PCR of the sample to be detected and the double fluorescent quantitative PCR of the standard plasmid are as follows: 30S at 95 ℃; 5S at 95 ℃, 34S at 60 ℃ and 40 cycles.
Preferably, the plasmid vector is a PZXK plasmid.
The invention has the beneficial effects that: the invention provides a primer group, a fluorescent probe group, a kit and a method for detecting the copy number of a CAR gene in an average single CAR-T cell; the method provided by the invention can accurately and effectively detect the copy number of the CAR gene in the T lymphocyte, and is simple to operate; the amplification peak of the primer group is single, and the specificity is good; the precise range of an amplification curve obtained by using the primer group, the fluorescent probe group and the detection method is 5-5 multiplied by 106Between copies, the detection range of the CAR gene copy number in a single CAR-T cell can reach 10 theoretically on average-6～106The result is accurate, and the detection range is wide. The primer set, the fluorescent probe set, the kit and the method provided by the invention can be applied to multiple aspects: in development, the transduction efficiency of the CAR gene can be detected; on quality release, the copy number of the CAR gene can be detected; clinically, changes in CAR-T cells can be detected; has very practical application significance.
Drawings
FIG. 1A is a structural map of the PZXK plasmid;
FIG. 1B is a structural map of the S1 plasmid;
FIG. 2 is a structural map of a standard plasmid;
FIG. 3A is a schematic representation of the CD3zeta-F/R primer dissolution curve;
FIG. 3B is a schematic representation of the CD3zeta-F/R primer amplification curve;
FIG. 3C is a schematic representation of the IFNG-F/R primer melting curve;
FIG. 3D is a schematic representation of the IFNG-F/R primer amplification curve;
FIG. 3E is a schematic diagram of the ABL-F/R primer melting curve;
FIG. 3F is a schematic diagram of an ABL-F/R primer amplification curve;
FIG. 3G is a schematic diagram of the melting curve of the WPRE-GJ-F/R primer;
FIG. 3H is a schematic diagram of the amplification curve of the WPRE-GJ-F/R primer;
FIG. 4A is a schematic representation of the CD3zeta-F/R primer amplification curve under the CD3 zeta-GJ-probe and ABL probe dual fluorescent probe amplification;
FIG. 4B is a schematic diagram of the ABL-F/R primer amplification curve under the CD3 zeta-GJ-probe and ABL probe dual fluorescent probe amplification;
FIG. 4C is a schematic diagram showing the amplification curve of the CD3zeta-F/R primer under the amplification of the CD3 zeta-GJ-probe and the IFNG probe dual fluorescent probe;
FIG. 4D is a schematic diagram showing the IFNG-F/R primer amplification curve under the amplification of the CD3 zeta-GJ-probe and the IFNG probe dual fluorescent probe;
FIG. 4E is a schematic diagram of a CD3zeta-F/R primer standard curve under the amplification of a CD3 zeta-GJ-probe and an IFNG probe dual fluorescent probe;
FIG. 4F is a schematic diagram of the IFNG-F/R primer standard curve under the amplification of the CD3 zeta-GJ-probe and the IFNG probe dual fluorescent probe;
FIG. 4G is a schematic diagram of the IFNG-F/R primer standard curve under single fluorescent probe amplification;
FIG. 4H is a schematic diagram of a CD3zeta-F/R primer standard curve under single fluorescent probe amplification.
Detailed Description
The invention provides a primer group for detecting the copy number of a CAR gene in an average single CAR-T cell, which comprises a primer pair for detecting the CAR gene and a primer pair for detecting an internal reference gene; the CAR gene comprises a CD3zeta gene and/or a WPRE gene, and the reference gene comprises an ABL gene and/or an IFNG gene; the nucleotide sequences of the primer pair for detecting the CD3zeta gene are shown as SEQ ID No.1 and SEQ ID No. 2; the nucleotide sequence of the primer pair for detecting the WPRE gene is shown as SEQ ID No.3 and SEQ ID No. 4; the nucleotide sequence of the primer pair for detecting the ABL gene is shown as SEQ ID No.5 and SEQ ID No. 6; the nucleotide sequence of the primer pair for detecting the IFNG gene is shown as SEQ ID No.7 and SEQ ID No. 8.
The invention provides a fluorescent probe set for detecting the copy number of a CAR gene in an average single CAR-T cell, which comprises a fluorescent probe for detecting the CAR gene and a fluorescent probe for detecting an internal reference gene; the CAR gene comprises a CD3zeta gene and/or a WPRE gene, and the reference gene comprises an ABL gene and/or an IFNG gene; the nucleotide sequence of the fluorescent probe for detecting the CD3zeta gene is shown as SEQ ID No. 9; the nucleotide sequence of the fluorescent probe for detecting the WPRE gene is shown as SEQ ID No. 10; the nucleotide sequence of the fluorescent probe for detecting the ABL gene is shown as SEQ ID No. 11; the nucleotide sequence of the fluorescent probe for detecting the IFNG gene is shown as SEQ ID No. 12.
Specific nucleotide sequences of the primer set and the fluorescent probe set involved in the present invention are shown in table 1.
TABLE 1 nucleotide sequences of primer sets and fluorescent probe sets
Figure BDA0002805893000000061
Figure BDA0002805893000000071
In the invention, a fluorescence reporter group is marked at the 5 'end of the fluorescent probe, and a fluorescence quenching group is marked at the 3' end of the fluorescent probe; in the present invention, the fluorescent reporter group is preferably one selected from FAM, Hex, VIC, ROX and Cy 5; the fluorescence quenching group is preferably one selected from BHQ1, TAMRA, JOE, BHQ2 and BHQ 3. The method for preparing the primer set and the fluorescent probe set is not particularly limited, and the primer set and the fluorescent probe set can be synthesized by a biotechnology company by adopting a conventional biosynthesis method in the field.
The invention also provides a kit for detecting the copy number of the CAR gene in an average single CAR-T cell, which comprises the primer group and the fluorescent probe group. In a preferred embodiment of the present invention, the kit comprises a primer pair for detecting the CD3zeta gene, a primer pair for detecting the IFNG gene, a fluorescent probe for detecting the CD3zeta gene and a fluorescent probe for detecting the IFNG gene.
In the present invention, the kit preferably further comprises a dual fluorescent quantitative PCR reaction reagent. In the invention, the dual fluorescent quantitative PCR reaction reagent comprises fluorescent quantitative PCR Mix, Dye II and water; the sources of the fluorescent quantitative PCR Mix and Dye II are not particularly limited in the present invention, and any commercially available product that is conventional in the art may be used. In the present invention, the water is preferably water dedicated for fluorescent quantitative PCR.
The invention provides a method of detecting the copy number of a CAR gene in an average single CAR-T cell, comprising the steps of:
1) constructing the CAR gene on a plasmid vector to obtain an S1 plasmid, and constructing the CAR gene and an internal reference gene on the plasmid vector together to obtain a standard plasmid; setting standard plasmids with different concentrations;
2) extracting the whole genome DNA of a sample to be detected, and adjusting the whole genome DNA to a proper concentration range of 1 ng/mul-500 ng/mul;
3) mixing the DNA to be detected, the primer group of the CAR gene, the primer group of the internal reference gene, the fluorescent probe of the CAR gene, the fluorescent probe of the internal reference gene and the dual fluorescent quantitative PCR reaction reagent in the step 2) to obtain a dual fluorescent quantitative PCR reaction system of a sample to be detected;
respectively mixing standard plasmids with different concentrations with a primer group of a CAR gene, a primer group of an internal reference gene, a fluorescent probe of the CAR gene, a fluorescent probe of the internal reference gene and a dual fluorescent quantitative PCR reaction reagent to obtain standard plasmid dual fluorescent quantitative PCR reaction systems with different concentrations;
performing fluorescent quantitative PCR detection on the sample dual-fluorescent quantitative PCR reaction system to be detected and the standard plasmid dual-fluorescent quantitative PCR reaction system with different concentrations on a computer to obtain the Ct value of the CAR gene of the sample to be detected, the Ct value of the reference gene of the sample to be detected, the Ct value of the CAR gene of the standard plasmid with different concentrations and the Ct value of the reference gene of the standard plasmid with different concentrations;
calculating the copy number of the CAR gene in the CAR-T cells of the sample to be detected;
CAR gene copy number in CAR-T cells of samples to be tested is 2-△△Ct×2；
Wherein the delta Ct is equal to the delta Ct-correction coefficient of the sample to be detected;
the delta Ct of the sample to be detected is the Ct value of the CAR gene of the sample to be detected-the Ct value of the reference gene of the sample to be detected;
the correction coefficient is (delta Ct1 +. DELTA Ct2 … …. delta. Ctn)/n;
where Δ Ctn is the Ctn value of the CAR gene of the standard plasmid-the Ctn value of the reference gene in the standard plasmid, and n is a specific concentration.
In the present invention, the plasmid vector is preferably a PZXK plasmid, and the map of the PZXK plasmid is shown in fig. 1A. In the invention, CAR gene and reference gene are jointly constructed on a plasmid vector to obtain a standard plasmid; setting standard plasmids with different concentrations; the function of the standard plasmid is to calibrate the difference of the amplification efficiency of the CAR gene and the internal reference gene within a certain concentration range. In the present invention, the concentration of the standard plasmid at different concentrations is preferably 1.25 copies/. mu.l to 1.25X 106copies/. mu.l; in the present invention, the standard plasmid is preferably diluted with water in a gradient, and the gradient dilution is preferably 10 times of the gradient dilution. The invention calculates the copy number of the standard plasmid with corresponding concentration according to the concentration and molecular weight of the standard plasmid, and the specific calculation formula is as follows: copy number 6.02 × 1023X nucleic acid concentration ÷ (DNA length x 660); copy number units are copies/ml, nucleic acid concentration units are g/ml, DNA length is 8399.
In the invention, the double fluorescent quantitative PCR reaction system of the sample to be detected is calculated by 20 μ l, and preferably comprises the following components:
Figure BDA0002805893000000091
the standard plasmid dual fluorescent quantitative PCR reaction system is calculated by 20 mu l, and preferably comprises the following components:
Figure BDA0002805893000000092
in the invention, the reaction procedures of the double fluorescence quantitative PCR of the sample to be detected and the double fluorescence quantitative PCR of the standard plasmid are as follows: 30S at 95 ℃; 5S at 95 ℃, 34S at 60 ℃ and 40 cycles.
After the double fluorescence quantitative PCR machine is used for fluorescence quantitative PCR detection, the CAR gene copy number in the CAR-T cell is calculated according to the detection result.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Specific nucleotide sequences of the primer set and the fluorescent probe set referred to in this example are shown in table 1. The design of the primers and probes in the embodiment is based on the gene sequence regions of the target gene CAR and the reference gene or the plasmid skeleton region PZXK, wherein the upstream primer CD3zeta-F is located in the CD3zeta signal region, and the downstream primer CD3zeta-R is located in the plasmid skeleton region. The structural map of the S1 plasmid of the present invention is shown in FIG. 1B by introducing the target gene CD3zeta into the plasmid PZXK, and the structural map of the standard plasmid of the present invention is shown in FIG. 2 by introducing the target gene CD3zeta and the reference gene into the plasmid PZXK.
Screening of primers and probes:
1) extracting the whole genome of untransfected T lymphocytes separated from PBMC of human peripheral blood, and adjusting the concentration to be 2.5 ng/mu l;
2) dilution with a 10-fold gradient of the genome in 1)S1 plasmid, with copy number ranging from 1.25 copies/. mu.l to 1.25X 106Between copies/. mu.l;
3) preparing SYBR reaction liquid, detecting on a machine, wherein a detection system and a detection program are shown in tables 2 and 3;
4) analyzing data, wherein the primer pairs CD3zeta-F/CD3-R, IFNG-F/IFNG-R and ABL-F/ABL-R have the highest specificity and the best sensitivity, and the dissolution curve and the amplification curve of the primers are shown in figures 3A-3H, so that the amplification peak is single and the amplification specificity is good;
5) standard plasmids were diluted with a 10-fold gradient of water to copy numbers ranging from 1.25 copies/. mu.l to 1.25X 106Between copies/. mu.l;
6) the reference gene primer and the fluorescent probe thereof, the target gene primer and the fluorescent probe thereof are configured in the same double-fluorescence reaction system, and are detected on the computer, wherein the detection system is shown as 4, and the detection results are shown as figures 4A-4H;
7) analyzing data, wherein the primer pair CD3zeta-F/CD3-R, IFNG-F/IFNG-R, CD3zeta-GJ fluorescent probe and IFNG fluorescent probe are combined into a dual fluorescent reaction system, the influence of each other is minimum, an amplification curve is consistent with a single fluorescent amplification curve, the standard curve equations of the primer pair CD3zeta-F/CD3-R are y which is 3.845x +11.337, R2 which is 0.9957, the standard curve equations of the primer pair IFNG-F/IFNG-R are y which is 3.8128x +11.406, R2 which is 0.9963, the correction coefficient CtDelta which is-0.05977 and almost zero; the reaction sensitivity is best, and the effective detection range is from 5copies to 5x 106Copying; the reaction specificity is highest, the amplification peak of the primer is single, and no non-specific amplification exists (FIG. 4A-FIG. 4H).
TABLE 2 preparation of primer specificity screening reaction solution
Mix 10μl
DyeII 0.4μl
Upstream primer 0.8μl
Upstream primer 0.8μl
Form panel 4μl
H2O 4μl
TABLE 3 primer screening reaction procedure
95℃ 30S
95℃ 5S
60℃ 34S
TABLE 4 Dual fluorescence reaction System
Mix 10μl
DyeII 0.2μl
Internal reference gene upstream primer 0.4μl
Internal reference gene downstream primer 0.4μl
Target gene upstream primer 0.4μl
Target gene downstream primer 0.4μl
Fluorescent probe for reference gene 0.8μl
Fluorescent probe for target gene 0.8μl
Form panel 4μl
H2O 2.6μl
Example 2
A method of detecting the copy number of a CAR gene in a T lymphocyte:
1) extracting genome from 8 samples to be detected, namely untransfected T cells, PZXK-transfected unloaded T cells and 6 batches of CAR-T cells transfected with anti-cMet CAR with different concentrations, using a blood genome DNA extraction kit (Tiangen, DP348), detecting concentration and purity, and diluting the genome into 2.5 ng/mu l by using water;
2) standard plasmids were diluted with a 10-fold gradient of water to copy numbers ranging from 1.25 copies/. mu.l to 1.25X 106Between copies/. mu.l, and a standard control plasmid was set at a concentration of 2.5X 103copies/μl；
3) Preparing double fluorescent reaction liquid, and detecting on a computer;
4) analyzing data, and subtracting the Ct value of the internal reference gene primer pair IFNG-F/IFNG-R from the Ct value of the target gene primer pair CD3zeta-F/CD3zeta-R to obtain a corresponding difference value delta Ct, wherein the average value of the delta Ct values of all gradients is the correction coefficient. Subtracting the Ct value of the reference gene of the sample to be detected from the Ct value of the target gene of the sample to be detected to obtain a delta Ct value of the sample to be detected, and subtracting the correction coefficient from the delta Ct value of the sample to be detected to obtain the delta Ct value, 2-△△CtThe value is the copy number of the human haploid genome, and the human genome is 2 times of that of the human haploid genome, 2-△△CtAnd 2 is the CAR gene copy number of the sample to be detected.
5) And substituting the Ct value of the sample to be detected into a calculation formula to obtain the corresponding copy number.
TABLE 5 Standard substance data analysis
Figure BDA0002805893000000121
TABLE 6 analysis of data of samples to be tested
Figure BDA0002805893000000122
Figure BDA0002805893000000131
The embodiment shows that the method provided by the invention can accurately and effectively detect the copy number of the CAR gene in the T lymphocyte, and has the advantages of simple operation, good specificity, accurate result and wide detection range.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (10)

1. A primer set for detecting the copy number of a CAR gene in an average single CAR-T cell, comprising a primer pair for detecting the CAR gene and a primer pair for an internal reference gene;
the CAR gene comprises a CD3zeta gene and/or a WPRE gene, and the reference gene comprises an ABL gene and/or an IFNG gene;
the nucleotide sequences of the primer pair for detecting the CD3zeta gene are shown as SEQ ID No.1 and SEQ ID No. 2;
the nucleotide sequence of the primer pair for detecting the WPRE gene is shown as SEQ ID No.3 and SEQ ID No. 4;
the nucleotide sequence of the primer pair for detecting the ABL gene is shown as SEQ ID No.5 and SEQ ID No. 6;
the nucleotide sequence of the primer pair for detecting the IFNG gene is shown as SEQ ID No.7 and SEQ ID No. 8.
2. A fluorescent probe set for detecting the copy number of a CAR gene in an average single CAR-T cell, comprising a fluorescent probe for detecting the CAR gene and a fluorescent probe for detecting an internal reference gene; the CAR gene comprises a CD3zeta gene and/or a WPRE gene, and the reference gene comprises an ABL gene and/or an IFNG gene;
the nucleotide sequence of the fluorescent probe for detecting the CD3zeta gene is shown as SEQ ID No. 9;
the nucleotide sequence of the fluorescent probe for detecting the WPRE gene is shown as SEQ ID No. 10;
the nucleotide sequence of the fluorescent probe for detecting the ABL gene is shown as SEQ ID No. 11;
the nucleotide sequence of the fluorescent probe for detecting the IFNG gene is shown as SEQ ID No. 12.
3. A kit for detecting the copy number of a CAR gene in an average individual CAR-T cell, comprising the primer set of claim 1 and the fluorescent probe set of claim 2.
4. The kit according to claim 3, comprising a primer set for detecting the CD3zeta gene, a primer set for detecting the IFNG gene, a fluorescent probe for detecting the CD3zeta gene and a fluorescent probe for detecting the IFNG gene.
5. The kit of claim 3 or 4, further comprising dual fluorescent quantitative PCR reaction reagents.
6. A method of detecting the copy number of a CAR gene in an average single CAR-T cell, comprising the steps of:
1) constructing the CAR gene and the reference gene on a plasmid vector together to obtain a standard plasmid; setting standard plasmids with different concentrations;
2) extracting the whole genome DNA of a sample to be detected, and adjusting the whole genome DNA to 1 ng/mu l-500 ng/mu l;
3) mixing the DNA to be detected, the primer group of the CAR gene, the primer group of the internal reference gene, the fluorescent probe of the CAR gene, the fluorescent probe of the internal reference gene and the dual fluorescent quantitative PCR reaction reagent in the step 2) to obtain a dual fluorescent quantitative PCR reaction system of a sample to be detected;
respectively mixing standard plasmids with different concentrations with a primer group of a CAR gene, a primer group of an internal reference gene, a fluorescent probe of the CAR gene, a fluorescent probe of the internal reference gene and a dual fluorescent quantitative PCR reaction reagent to obtain standard plasmid dual fluorescent quantitative PCR reaction systems with different concentrations;
performing fluorescent quantitative PCR detection on the sample dual-fluorescent quantitative PCR reaction system to be detected and the standard plasmid dual-fluorescent quantitative PCR reaction system with different concentrations on a computer to obtain the Ct value of the CAR gene of the sample to be detected, the Ct value of the reference gene of the sample to be detected, the Ct value of the CAR gene of the standard plasmid with different concentrations and the Ct value of the reference gene of the standard plasmid with different concentrations;
calculating the copy number of the CAR gene in the CAR-T cells of the sample to be detected;
CAR gene copy number in CAR-T cells of samples to be tested is 2-△△Ct×2；
Wherein the delta Ct is equal to the delta Ct-correction coefficient of the sample to be detected;
the delta Ct of the sample to be detected is the Ct value of the CAR gene of the sample to be detected-the Ct value of the reference gene of the sample to be detected;
the correction coefficient is (delta Ct1 +. DELTA Ct2 … …. delta. Ctn)/n;
where Δ Ctn is the Ctn value of the CAR gene of the standard plasmid-the Ctn value of the reference gene in the standard plasmid, and n is a specific concentration.
7. The method according to claim 6, wherein the concentration of the standard plasmid at different concentrations is 1.25copies/μ l to 1.25X 106copies/μl。
8. The method according to claim 6, wherein the sample to be detected comprises the following components in 20 μ l:
Figure FDA0002805892990000031
the standard plasmid dual-fluorescence quantitative PCR reaction system is calculated by 20 mu l and comprises the following components:
Figure FDA0002805892990000032
9. the method of claim 6, wherein the reaction procedures of the double fluorescence quantitative PCR and the standard plasmid double fluorescence quantitative PCR of the sample to be detected are as follows: 30S at 95 ℃; 5S at 95 ℃, 34S at 60 ℃ and 40 cycles.
10. The method of claim 6, wherein the plasmid vector is a PZXK plasmid.
CN202011370271.8A 2020-11-30 2020-11-30 Primer set, fluorescent probe set, kit and method for detecting copy number of CAR gene in average single CAR-T cell Pending CN112481361A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114686576A (en) * 2022-04-18 2022-07-01 武汉科技大学 Method for detecting copy number of lentiviral vector of single CAR-T cell and application of method
CN116356005A (en) * 2023-04-28 2023-06-30 宁波熙宁检测技术有限公司 Composition for detecting CAR-T cell copy number and application thereof
WO2024046474A1 (en) * 2022-09-01 2024-03-07 南京传奇生物科技有限公司 Method for detecting car copy number

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107188968A (en) * 2017-05-08 2017-09-22 上海神因生物科技有限公司 A kind of people source Chimeric antigen receptor of targeting PDPN genes and application thereof
CN109722472A (en) * 2019-02-28 2019-05-07 上海邦耀生物科技有限公司 Detect the universal PCR primer combination and application of Chimeric antigen receptor gene
CN109971836A (en) * 2017-12-28 2019-07-05 上海细胞治疗研究院 The method and kit of double fluorescent quantitative PCR measurement CAR copy number
WO2020028533A1 (en) * 2018-08-01 2020-02-06 Yale University Compositions and methods for identification of membrane targets for enhancement of t cell activity against cancer
CN110863051A (en) * 2019-12-13 2020-03-06 广州迈景基因医学科技有限公司 Primer, system and kit for MET gene amplification detection
CN111334565A (en) * 2020-03-31 2020-06-26 时力生物科技（北京）有限公司 Method for detecting CAR gene copy number in T lymphocyte by fluorescent quantitative PCR
CN111909931A (en) * 2019-05-08 2020-11-10 深圳宾德生物技术有限公司 Fluorescent probe, primer pair, fluorescent quantitative PCR kit and detection method

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107188968A (en) * 2017-05-08 2017-09-22 上海神因生物科技有限公司 A kind of people source Chimeric antigen receptor of targeting PDPN genes and application thereof
CN109971836A (en) * 2017-12-28 2019-07-05 上海细胞治疗研究院 The method and kit of double fluorescent quantitative PCR measurement CAR copy number
WO2020028533A1 (en) * 2018-08-01 2020-02-06 Yale University Compositions and methods for identification of membrane targets for enhancement of t cell activity against cancer
CN109722472A (en) * 2019-02-28 2019-05-07 上海邦耀生物科技有限公司 Detect the universal PCR primer combination and application of Chimeric antigen receptor gene
CN111909931A (en) * 2019-05-08 2020-11-10 深圳宾德生物技术有限公司 Fluorescent probe, primer pair, fluorescent quantitative PCR kit and detection method
CN110863051A (en) * 2019-12-13 2020-03-06 广州迈景基因医学科技有限公司 Primer, system and kit for MET gene amplification detection
CN111334565A (en) * 2020-03-31 2020-06-26 时力生物科技（北京）有限公司 Method for detecting CAR gene copy number in T lymphocyte by fluorescent quantitative PCR

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ALEXANDER KUNZ等: "Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients", 《MOLECULAR THERAPY: METHODS & CLINICAL DEVELOPMENT》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114686576A (en) * 2022-04-18 2022-07-01 武汉科技大学 Method for detecting copy number of lentiviral vector of single CAR-T cell and application of method
WO2024046474A1 (en) * 2022-09-01 2024-03-07 南京传奇生物科技有限公司 Method for detecting car copy number
CN116356005A (en) * 2023-04-28 2023-06-30 宁波熙宁检测技术有限公司 Composition for detecting CAR-T cell copy number and application thereof
CN116356005B (en) * 2023-04-28 2023-09-26 宁波熙宁检测技术有限公司 Composition for detecting CAR-T cell copy number and application thereof

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