Method for detecting copy number of CLDN18.2 CAR in body tissue or blood DNA by using fluorescent probe

Abstract

The invention discloses a method for detecting the copy number of CLDN18.2 CAR in body tissue or blood DNA through a fluorescent probe. The method comprises the steps that a tissue or blood DNA sample is obtained; mixing the amplification system with an organism tissue or blood DNA sample, and carrying out isothermal amplification reaction to obtain an amplification product; in the isothermal amplification reaction process, a fluorescent probe is used for detecting an amplification product in real time so as to judge whether a fluorescent signal is amplified or not, and if yes, it is indicated that CLDN18.2 CAR copy exists. According to the detection method, the copy number of the CLDN18.2 CAR is quantified through the fluorescence intensity of the probe, the minimum detection concentration can reach 10 copies / microliter, and the copy number of the CLDN18.2 CAR in body tissue or blood can be successfully detected.

Description

Technical Field

[0001] The present invention relates to molecular biological detection, and in particular to a method for detecting the copy number of CLDN18.2 CAR in body tissue or blood DNA using a fluorescent probe. Background Art

[0002] CAR generally has a scFv single-chain antibody (the variable regions of the antibody light and heavy chains are linked by a linker polypeptide and are responsible for binding to antigens), which is connected to the transmembrane region and intracellular signaling structure through a hinge structure, in which the intracellular signaling domain is responsible for transmitting T cell activation and activation signals to the cell. T cells modified by the CAR gene can recognize specific antigen molecules on the tumor cell membrane and activate the T cell's killing, proliferation, and cytokine secretion capabilities.

[0003] Claudin18.2 belongs to the Claudin protein family. The Claudin family is a class of integrin membrane proteins present in the tight junctions of epithelial and endothelial cells. Currently, there are 27 members of the Claudin family in mammals. Among them, Claudin-18 splice variant 2 (CLDN18.2) is a gastric-specific tight junction protein that is expressed in many cancers. The CLDN18.2 gene is abnormally activated and overexpressed in a variety of primary malignant tumors such as breast cancer, colon cancer, liver cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer, especially in digestive system malignancies, including gastric cancer (70%), pancreatic cancer (50%), esophageal cancer (30%), etc. In addition, in tumor tissues, Claudin18.2 is also involved in the proliferation, differentiation, and migration of tumor cells. The characteristics of Claudin18.2 (highly selective, widely expressed only in cancer cells) make it an ideal tumor immune target. Currently, the research on targeting Claudin18.2 is mainly focused on gastric cancer. Recently, its expression in pancreatic cancer (50%), esophageal cancer and lung cancer has been found, which also shows the potential for diagnosis and treatment of other tumors.

[0004] The Patsnap database shows that there are 28 CAR-T drugs under development for CLDN18.2. Currently, the main method for detecting the tissue distribution of CAR-T is immunohistochemistry, but this method requires the development of antibodies that specifically recognize scFv, and has low sensitivity, high background, and long detection cycle. These shortcomings bring great inconvenience to the safety evaluation of CAR-T. Therefore, it is particularly important to develop a CAR-T detection method with short detection cycle, high sensitivity, and low cost. Summary of the invention

[0005] Based on the above problems, the problem to be solved by the present invention is to provide a method for detecting the copy number of CLDN18.2 CAR in body tissue or blood DNA using a fluorescent probe with a short detection cycle, high sensitivity and low cost.

[0006] The technical solution of the present invention is as follows:

[0007] A method for detecting the copy number of CLDN18.2 CAR in body tissue or blood DNA using a fluorescent probe comprises the following steps:

[0008] Obtain DNA samples from body tissue or blood;

[0009] The amplification system is mixed with a body tissue or blood DNA sample to perform an isothermal amplification reaction to obtain an amplification product;

[0010] During the isothermal amplification reaction, the amplified product is detected in real time using a fluorescent probe to determine whether there is amplification of the fluorescent signal, which indicates the presence of CLDN18.2 CAR copies;

[0011] The components of the amplification system include: 5-30 μl PCR reaction solution, 0.5-2 μl fluorescent probe and 0.5-2 μl CLDN18.2 CAR copy-specific primer.

[0012] In the detection method, the volume of the body tissue or blood DNA sample is 1 to 10 μl and the concentration is 100 to 500 ng / μl.

[0013] In the detection method, in the isothermal amplification reaction, the reaction temperature is 58° C. to 95° C. and the reaction time is 90 min to 95 min.

[0014] In the detection method, the isothermal amplification reaction also includes the following reaction steps and reaction conditions:

[0015] Pre-denaturation: 95°C, 10 min;

[0016] Denaturation: 95°C, 15s;

[0017] Replication: 59°C, 59.5°C, 60°C, 60.5°C, or 61°C, 1 min;

[0018] The denaturation step and the replication step are performed alternately for 40 cycles.

[0019] In the detection method, the CLDN18.2 CAR copy-specific primers include a forward primer and a reverse primer; the forward primer is CLDN18.2-scFv-05-F, and the reverse primer is CLDN18.2-scFv-05-R; wherein the nucleic acid sequence of the CLDN18.2-scFv-05-F is: 5'-GCTGATCTACTGGGCTAG-3';

[0020] The nucleic acid sequence of CLDN18.2-scFv-05-R is: 5'-TAGGCGTTCTGACAGTAG-3'.

[0021] In the detection method, the fluorescent probe is CLDN18.2 scFv-05-probe; wherein the nucleic acid sequence of the CLDN18.2 scFv-05-probe is: 5'-TGCTAATGGTCAGGGTGAAGTCGG-3'.

[0022] In the detection method, the fluorescent detection probe is a stem-loop structure and has a fluorescent group labeled at the 5' end and a quenching group labeled at the 3' end.

[0023] In the detection method, a second quenching group is added in the middle of the stem-loop structure.

[0024] The detection method for detecting the copy number of CLDN18.2 CAR in body tissue or blood provided by the present invention adopts an amplification system to mix with the body tissue or blood DNA sample to perform a two-step amplification cycle; during the amplification process, the amplification product is detected in real time by a fluorescent probe, and the copy number of CLDN18.2 CAR is quantified by the fluorescence intensity of the probe, and its minimum detection concentration can reach 10 copies / μl, and the copy number of CLDN18.2 CAR in body tissue or blood can be successfully detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a detection curve diagram of the standard double-quenching probe prepared in Example 1. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0027] The method for detecting the copy number of CLDN18.2 CAR in tissue or blood by using a fluorescent probe provided by the present invention comprises the following steps: mixing the amplification system with tissue or blood DNA, and performing two-step isothermal amplification; during the amplification process, using a fluorescent probe method to perform real-time detection of the amplification product, and quantifying the copy number of CAR by fluorescence intensity.

[0028] The specific detection process steps are as follows:

[0029] S1. Obtain DNA samples from body tissue or blood;

[0030] S2, mixing the amplification system with the body tissue or blood DNA sample, performing an isothermal amplification reaction, and obtaining an amplification product;

[0031] S3, during the isothermal amplification reaction, using a fluorescent probe to detect the amplification product in real time;

[0032] S4. Determine whether there is fluorescence signal amplification (which can be judged as positive); if so, it indicates that CLDN18.2 CAR copies exist; otherwise, it indicates that there are no CLDN18.2 CAR copies.

[0033] In step S1 of the above detection method, the volume of the body tissue or blood DNA sample is 1-10 μl and the concentration is 100-500 ng / μl; wherein the tissue or blood DNA sample can be derived from a healthy or patient human body, or from an animal.

[0034] In step S2 of the above detection method, the amplification system includes the following components:

[0035] 5-30 μl PCR reaction solution;

[0036] 0.5-2 μl fluorescent probe;

[0037] 0.5-2 μl CLDN18.2 CAR copy-specific primers;

[0038] Among them, the PCR reaction solution is 2×Mix (commercially available); the CLDN18.2 CAR copy-specific primers include forward primer CLDN18.2-scFv-05-F and reverse primer CLDN18.2-scFv-05-R sequences, and the amount of each is 0.5 to 2 μl; the fluorescent probe is CLDN18.2 scFv-05-probe.

[0039] The PCR reaction solution may also include 5 to 15 μl of ddH2O (sterile water) and 2 to 10 μl of PCR standard or DNA sample to be detected; wherein ddH2O is used to prepare the working concentration of the fluorescent probe and specific primers.

[0040] The CLDN18.2 CAR copy-specific primers consist of a forward primer and a reverse primer:

[0041] Forward primer CLDN18.2-scFv-05-F sequence: 5′-GCTGATCTACTGGGCTAG-3′;

[0042] Reverse primer CLDN18.2-scFv-05-R sequence: 5′-TAGGCGTTCTGACAGTAG-3′.

[0043] The sequence of the fluorescent probe CLDN18.2 scFv-05-probe is: 5'-TGCTAATGGTCAGGGTGAAGTCGG-3'; the fluorescent detection probe is a stem-loop structure and is labeled with a fluorescent group at the 5' end and a quenching group at the 3' end, and a second quenching group may be added in between.

[0044] In step S2, in the above isothermal amplification reaction, the reaction temperature is 59°C to 95°C, and the reaction time is 90min to 95min.

[0045] Preferably, the isothermal amplification reaction further comprises the following reaction steps and reaction conditions:

[0046] Pre-denaturation: 95°C, 10 min;

[0047] Denaturation: 95°C, 15s;

[0048] Replication: 59°C, 59.5°C, 60°C, 60.5°C, or 61°C, 1 min;

[0049] The denaturation and replication steps need to be performed alternately for 40 cycles.

[0050] The technical solution of the present invention is further described in detail below through specific embodiments.

[0051] 1. Solution Design

[0052] 1. Establishment of standard curves of two fluorescent probes;

[0053] 2. Extraction of DNA from mouse tumor tissue and spleen;

[0054] 3. Two fluorescent probes were used to detect the CAR copy number in the tumor tissue and spleen of mice infused with CAR-T cells. 2. Specific embodiments

[0056] Example 1 Establishment of fluorescent probe Q-PCR standard curve

[0057] 1.1. Prepare the required reagents and consumables, spray them with 75% alcohol and place them in a biosafety cabinet. Keep them away from light during the experimental operation;

[0058] 1.2. Perform gradient dilution of the standard. Add 5 μl of the standard to 45 μl of sterile water and dilute 10-fold in sequence to obtain 1×10 7 , 1×10 6 , 1×10 5 , 1×104 , 1×10 3 , 1×10 2 , 1×10, 0 gradient (ie, ddH2O) solution; 1.3, using sterile water to dilute the fluorescent probe 1 10 times to prepare a probe with a working concentration of 10 nM;

[0059] 1.4. Prepare PCR reaction solution according to the reaction system in Table 1 below and mix well.

[0060] Table 1. Single tube reaction system for standard products

[0061]

[0062] 1.5. Take 5 μl of the diluted standard sample and add it to the eight tube strips. Add 25 μl of the PCR reaction solution to each tube and mix well. Wait for the machine to start, set the following reaction program and run:

[0063] S1.1, pre-denaturation: 95℃, 10min;

[0064] S1.2, denaturation: 95°C, 15s;

[0065] S1.3, replication: 59°C, 59.5°C, 60°C, 60.5°C or 61°C, 1 min;

[0066] The denaturation step and the replication step need to be performed alternately for 40 cycles.

[0067] The above test results are as follows Figure 1 As shown in Table 2, the amplification efficiency of CLDN18.2-scFv-05-Probe at an annealing temperature of 59°C was 98.7%, the amplification efficiency at an annealing temperature of 59.5°C was 88.88%, the amplification efficiency at an annealing temperature of 60°C was 86.55%, the amplification efficiency at an annealing temperature of 60.5°C was 83.31%, and the amplification efficiency at an annealing temperature of 61°C was 96.189%. Therefore, when the CLDN18.2-scFv-05-Probe fluorescent probe detects a sample, an annealing temperature of 59°C is preferred.

[0068] Table 2 Average Ct values ​​of standard products under different annealing temperature conditions

[0069]

[0070] exist Figure 1 The annealing temperature is T m Indicates; among them, the five figures a, b, c, d, and e correspond to the double quenching probe detection curves at 59°C, 59.5°C, 60°C, 60.5°C, and 61°C, respectively.

[0071] Example 2 Extraction of mouse tissue DNA

[0072] DNA from mouse tumor tissues and spleen was extracted using a genomic DNA extraction kit.

[0073] 2.1. Weigh a certain amount of animal tissue, of which spleen tissue is ≤10 mg and tumor tissue is ≤25 mg. Use a scalpel to chop the tissue into a homogenate and resuspend it in 1 ml of serum-free DMEM medium.

[0074] 2.2, centrifuge at 11200g for 1 min, pour off the supernatant, add 200 μl buffer GA, and vortex until completely suspended;

[0075] 2.3. Add 20 μl of Proteinase K solution and mix well; place at 56°C until the tissue is dissolved, centrifuge briefly to remove the water droplets on the inner wall of the tube cap, and then proceed to the next step. Different tissues have different lysis times, usually 1-3 hours to complete;

[0076] 2.4. Add 200 μl of buffer GB, mix thoroughly by inversion, and place at 70°C for 10 min. The solution should become clear. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0077] 2.5. Add 200 μl of anhydrous ethanol and vortex thoroughly for 15 seconds. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0078] 2.6. Add the solution and flocculent precipitate obtained in the previous step to the adsorption column CB3, centrifuge at 13400g for 30s, discard the waste liquid, and put the adsorption column CB3 back into the collection tube;

[0079] 2.7. Add 500 μl of buffer GD to the adsorption column CB3, centrifuge at 13400 g for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube;

[0080] 2.8. Add 600 μl of rinse solution PW to the adsorption column CB3, centrifuge at 13400 g for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube;

[0081] 2.9. Repeat the above step 2.8;

[0082] 2.10. Place the adsorption column CB3 back into the collection tube, centrifuge at 13400g for 2 minutes, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material;

[0083] 2.11. Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μl of elution buffer TE to the middle part of the adsorption membrane, let it stand at room temperature for 2-5 minutes, centrifuge at 13400g for 2 minutes, and collect the solution into the centrifuge tube.

[0084] The test results are shown in Table 3 below. The concentration of DNA extracted from the tumor tissues and kidneys of each mouse was in the range of 95.0-221.8 ng / ml, and the A260 / 280 ratio was in the range of 1.8-2.0, indicating that the quality of the extracted DNA met the PCR requirements.

[0085] Table 3. DNA concentration in mouse tumors and kidneys

[0086]

[0087] Example 3 Detection of CAR copy number in tumor tissue and spleen by fluorescent probe method

[0088] 3.1. Prepare the required reagents and consumables, spray them with 75% alcohol and place them in the biosafety cabinet. Do not turn on the light during the experimental operation to keep away from light;

[0089] 3.2. Perform gradient dilution of the standard. Add 5 μl of the standard to 45 μl of sterile water and dilute by 10 times to obtain 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 of solution;

[0090] 3.3. Dilute the fluorescent probe 10 times with sterile water to prepare a probe with a working concentration of 10 nM;

[0091] 3.4. Prepare the mixed solution according to the reaction system in the table below and mix well. Note that the standard sample is not included for the time being. The standard sample requires 8 wells according to the gradient and needs to be repeated, for a total of 16 wells;

[0092] Table 4. Single tube reaction system for standards.

[0093]

[0094] 3.5. Take 5 μl of the diluted standard sample and add it to the eight-tube strips. Add 25 μl of the mixed solution to the eight-tube strips, mix well, and wait for loading.

[0095] 3.6. Prepare the PCR reaction solution in the sample tube according to the configuration method in the table below, and add 300ng of mouse tissue DNA sample to each tube, mix well, and load onto the machine;

[0096] Table 5. Sample single tube reaction system

[0097]

[0098] 3.8. Set up the following reaction program and run the fluorescence quantitative PCR instrument. Set up the following reaction program and run:

[0099] S3.1, pre-denaturation: 95℃, 10min;

[0100] S3.2, denaturation: 95°C, 15s;

[0101] S3.3, replication: 59°C, 1 min;

[0102] The denaturation step and the replication step need to be performed alternately for 40 cycles.

[0103] The results of the on-machine detection are shown in Table 6 below. The Ct value of each mouse was around 30, the CAR copy number was 2.75-2.9, and the CAR copy number in the tumor tissue of the group of CLDN18.2 CAR-T cells was significantly higher than that of the NT group (control group). There was no significant difference in the CLDN18.2 CAR copy number in the kidney between the NT and CAR-T groups. The test showed that the fluorescent probe method can be used for CAR copy number detection in tumor tissue samples with good specificity.

[0104] Table 6. Average Ct values ​​in mouse tumors and kidneys

[0105]

[0106] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0107] The above-mentioned embodiment only expresses one implementation mode of the present invention, and its description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A method for detecting the copy number of CLDN18.2 CAR in body tissue or blood DNA using a fluorescent probe, characterized in that: The steps include: Obtain DNA samples from body tissue or blood; The amplification system is mixed with a body tissue or blood DNA sample to perform an isothermal amplification reaction to obtain an amplification product; During the isothermal amplification reaction, the amplification product is detected in real time using a fluorescent probe to determine whether there is amplification of the fluorescent signal; If yes, it indicates the presence of CLDN18.2 CAR copies; The components of the amplification system include: 5-30 μl PCR reaction solution, 0.5-2 μl fluorescent probe and 0.5-2 μl CLDN18.2 CAR copy-specific primer.

2. The detection method according to claim 1, characterized in that: The volume of the body tissue or blood DNA sample is 1-10 μl and the concentration is 100-500 ng / μl.

3. The detection method according to claim 1, characterized in that: In the isothermal amplification reaction, the reaction temperature is 58° C. to 95° C., and the reaction time is 90 min to 95 min.

4. The detection method according to claim 3, characterized in that: The isothermal amplification reaction also includes the following reaction steps and reaction conditions: Pre-denaturation: 95°C, 10 min; Denaturation: 95°C, 15s; Replication: 59°C, 59.5°C, 60°C, 60.5°C, or 61°C, 1 min; The denaturation step and the replication step are performed alternately for 40 cycles.

5. The detection method according to claim 1, characterized in that: The CLDN18.2 CAR copy-specific primers include a forward primer and a reverse primer; the forward primer is CLDN18.2-scFv-05-F, and the reverse primer is CLDN18.2-scFv-05-R.

6. The detection method according to claim 5, characterized in that: The nucleic acid sequence of the CLDN18.2-scFv-05-F is: 5'-GCTGATCTACTGGGCTAG-3'; the nucleic acid sequence of the CLDN18.2-scFv-05-R is: 5'-TAGGCGTTCTGACAGTAG-3'.

7. The detection method according to claim 1, characterized in that: The fluorescent probe is CLDN18.2scFv-05-probe.

8. The detection method according to claim 7, characterized in that: The nucleic acid sequence of the CLDN18.2 scFv-05-probe is: 5'-TGCTAATGGTCAGGGTGAAGTCGG-3'.

9. The detection method according to claim 1, characterized in that: The fluorescent detection probe is a stem-loop structure, and the 5' end is labeled with a fluorescent group and the 3' end is labeled with a quenching group.

10. The detection method according to claim 9, characterized in that: A second quenching group is added in the middle of the stem-loop structure.

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