CN110564858A - RT-PCR kit for predicting curative effect of immunodetection point regulation type medicines and prediction method thereof - Google Patents
RT-PCR kit for predicting curative effect of immunodetection point regulation type medicines and prediction method thereof Download PDFInfo
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Abstract
The invention discloses an RT-PCR kit for predicting curative effect of immunodetection point regulation medicines and a prediction method thereof. The kit comprises a primer for RT-PCR amplification and auxiliary components thereof; the primers comprise a primary RT-PCR amplification primer and a specific primer for carrying out secondary amplification on a TCR region of a primary amplification product; the first-round RT-PCR amplification primer is shown as SEQ ID NO. 1-40; the second round amplification primer is shown as SEQ ID NO. 41-50. The invention is based on high-throughput sequencing technology and target region specificity amplification technology, separates lymphocytes from peripheral blood of a subject, extracts RNA, carries out reverse transcription to obtain cDNA, amplifies a TCR variable region by using a specificity primer to obtain sequence information of the TCR variable region, and evaluates the diversity of the TCR variable region. The method realizes the prediction and evaluation of the curative effect of the immunodetection point regulation type medicament at the gene molecule level by using a small amount of peripheral blood samples.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to an RT-PCR kit for predicting curative effects of immunodetection point regulation medicines and a prediction method thereof.
Background
An important function of the immune system is to distinguish autologous normal cells from foreign/abnormal cells, thereby attacking the foreign/abnormal cells while retaining normal cells. To avoid accidental injury, T cells in humans, when activated, express certain proteins that function as "immunodetection points", such as PD1 and CTLA 4. If the surface of a certain cell has matched protein, the T cell can be released from attacking the cell, for example, PD-L1/PD-L2 binds to PD-1, CD80/CD86 binds to CTLA-4. Certain cancer cells may express such a matching signal protein, thereby avoiding attack by the immune system.
The immunodetection point regulating medicine can block the combination of the detected cancer cell surface and its ligand protein to eliminate the immune escape of cancer cell. Various immunodetection point modulating drugs are currently on the market, such as Pembroliz μ Mab targeting PD-1 (Keytruda), Nivol μ Mab, ateuzoliz μ Mab targeting PD-L1 (tecentiq), Avel μ Mab (Bavencio), Durval μ Mab (infinzi), and ipilimumab ipiim μ Mab targeting CTLA-4 (Yervoy).
In order to predict the curative effect of immunodetection point-regulated drugs on tumors, the existing methods are (taking PD-1 as an example):
1. Tumor tissue sections were obtained, and the expression level of PD-L1 on the surface of cancer cells was detected by immunohistochemical method. The drawbacks of this method are: 1) puncture or minimally invasive surgery is needed to obtain tumor tissues, and the risk of tumor spread is increased when some patients do not want to cooperate; 2) the response of the patient's immune system to drugs cannot be predicted, so even if cancer cells express PD-L1, immunodetection point-modulating drugs are effective in only about 20% of patients, and the prediction accuracy is not high.
2. tumor tissue sections were taken and tested for DNA mismatch repair deficiency (dMMR) using immunohistochemical methods, or microsatellite instability (MSI) or Tumor Mutational Burden (TMB) using genetic assays. The drawbacks of this method are: 1) puncture or minimally invasive surgery must be performed; 2) it is unpredictable whether the immune system of a patient responds to tumor mutations, so even if the detection values of dMMR, MSI or TMB are high, immunodetection point regulation medicines are effective for a few patients, and the prediction accuracy is not high.
3. tumor tissue sections were taken and immunohistochemical methods were used to detect the number and type of lymphocytes infiltrated in the tumor tissue. The drawbacks of this method are: 1) puncture or minimally invasive surgery must be performed; 2) the degree of response of the immune system of a patient to tumors cannot be predicted, so even if a high number of tumor infiltrating lymphocytes are detected and PD-1 expression is detected, the immunodetection point regulation type medicine is only effective for a few patients, and the prediction accuracy is not high.
4. Blood from the patient was collected for genetic testing to detect circulating tumor DNA (ctDAN) and Circulating Tumor Cells (CTC). The drawbacks of this method are: the degree of the response of the immune system of a patient to the tumor cannot be predicted, ctDNA and CTC only reflect the progress of the tumor at that time, and the accuracy of predicting the curative effect of immunodetection point regulation medicines is not high.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the RT-PCR kit for predicting the curative effect of the immunodetection point regulation type medicine and the prediction method thereof, and can effectively solve the problem that the prior method can not predict the curative effect of the immunodetection point regulation type medicine on tumors.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
an RT-PCR kit for predicting the curative effect of immunodetection point regulation medicines comprises a primer for RT-PCR amplification and auxiliary components thereof; the primers comprise a primary RT-PCR amplification primer and a specific primer for performing secondary PCR amplification on a TCR region of a primary amplification product;
The first-round RT-PCR amplification primer is shown as SEQ ID NO. 1-40; the specific primers for the second round of PCR amplification are shown as SEQ ID NO. 41-50.
Further, the auxiliary components comprise RT-PCR buffer solution and MgCl2、dNTP、ddH2O, and an enzyme.
The method for detecting the curative effect of the immunodetection point regulation type medicine by adopting the kit comprises the following steps:
(1) Extracting RNA of a sample to be detected after the medicine is applied;
(2) Carrying out reverse transcription on an RNA template of a sample to be detected, then carrying out first-round RT-PCR amplification on cDNA prepared by the reverse transcription, and collecting a purified amplification product;
(3) Performing second round of PCR amplification on the amplification product obtained in the step (2) again to amplify the TCR variable region to obtain a variable region sequence;
(4) Calculating D based on the variable region sequence obtained in step (3)50the value is obtained.
Further, D in the step (4)50The values are calculated as follows: sequencing to obtain TCR variable region sequence information, comparing to determine the total number of variable region sequences as N, the number of types of sequences different from variable region genes as C, the copy number of each sequence is N1 and N2 … … NC, sequencing the sequences according to the copy number from at least N1 and N2 … … NC, the number of clone types with the most copy number and occupying 50 percent of the total number of functional variable region sequences is H, and the ratio of H to C is D50。
Further, when D50<when 0.01 hour, the immunity of the patient is low, and the remarkable curative effect is difficult to obtain by using the immunodetection point regulating medicines;
When D is more than or equal to 0.0150Less than or equal to 0.015, which indicates that the immunity of the patient is poor, and the use of the immunodetection point regulation medicine can possibly achieve certain effect;
When 0.015<D50The method shows that the immunity of the patient is relatively good, and the use of the immunodetection point regulation medicine has a great possibility of obtaining a remarkable effect.
Wherein, the definition of the curative effect of the immunodetection point regulation type medicine is as follows:
Complete remission (Complete Release, CR): all target lesions disappeared, no new ones appeared, and tumor markers were normal for at least 4 weeks.
Partial remission (Complete Release, CR): the sum of the maximum diameters of the target lesions is reduced by more than or equal to 30 percent and is maintained for at least 4 weeks.
Stable Disease (SD): the sum of the maximum diameters of the target lesions reduces the underreached PR or increases the underreached PD.
Disease Progression (PD): the sum of the maximum diameters of the target lesions is at least increased by more than or equal to 20 percent, or new lesions appear.
Further, TCR variable region complementarity determining region 3(CDR3) was used to determine TCR variable region sequence uniqueness.
The invention has the beneficial effects that:
The invention is based on high-throughput sequencing technology and target region specificity amplification technology, separates lymphocytes from peripheral blood of a subject, extracts RNA, carries out reverse transcription to obtain cDNA, amplifies a TCR variable region by using a specificity primer to obtain sequence information of the TCR variable region, and evaluates the diversity of the TCR variable region. The method realizes the prediction and evaluation of the curative effect of the immunodetection point regulation type medicament at the gene molecule level by using a small amount of peripheral blood samples.
Drawings
FIG. 1 shows RNA extraction quality test;
FIG. 2 shows the quality of amplification of the TCR variable region;
FIG. 3 shows the therapeutic effect of PD1 inhibitor on subject D50A distribution relationship;
FIG. 4 is a graph according to subject D50Predicting the efficacy of PD1 inhibitor.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
Example 1 construction of method for immune profiling and predicting the efficacy of immunodetection point-modulating drugs
First, lymphocyte separation and mRNA extraction
1. Lymphocyte isolation
Taking 10mL of venous blood of 6 subjects, placing the venous blood into an ultraviolet cover EDTA anticoagulation tube, and separating lymphocytes by using human lymphocyte separation liquid (Tianjin third level positive, the product number LTS1077), wherein the specific operation steps are as follows:
(1) 10mL of collected fresh blood is added into a 50mL centrifuge tube, physiological saline is added to the centrifuge tube to reach a constant volume of 25mL, and the mixture is gently blown and uniformly mixed.
(2) in a new 50mL centrifuge tube, 25mL of lymphocyte separation medium (equivalent) was added, and then the blood sample was pipetted gently onto the surface of the medium along the wall, at 500g, 20 ℃, and centrifuged for 30min at an increase rate of 2 and a decrease rate of 1(thermo low speed centrifuge).
(3) after centrifugation, a large part of the upper layer separation solution is carefully sucked off by a 10mL pipette, then the second layer of the annular milky white lymphocyte layer is sucked into a new 50mL centrifuge tube, physiological saline is added to the centrifuge tube to reach a constant volume of 50mL, the mixture is gently inverted and mixed, 300g is centrifuged at 20 ℃ for 8min, and the supernatant is discarded.
(4) and (3) cleaning the cells again, firstly, scattering the cells at the bottom of the tube, then adding physiological saline to a constant volume of 20mL, slightly reversing the mixture up and down, mixing the mixture evenly, centrifuging the mixture for 5min at the temperature of 20 ℃, and removing the supernatant.
(5) The homogeneous suspension cells were gently pipetted by adding 2mL of PBS to a 50mL centrifuge tube to obtain isolated lymphocytes.
(6) Cell counting: diluting 100 μ L of the suspension with 900 μ L of PBS 10 times, mixing, and counting cells (the dilution can be adjusted according to the cell amount), wherein the cell counting formula is as follows: cell number density 4 large lattice cell total number × dilution multiple × 104And/4 equals cell number/mL.
2. Extraction and detection of lymphocyte genome mRNA
the procedure was as described for Trizol reagent (Ambion, cat. No. 15596026), typically 5X 106～1×107The number of cells is strictly counted, and the cells are not suitable to be too many, so that the RNA enzyme is not completely inhibited, and the RNA is degraded. The specific operation steps are as follows:
(1) the isolated suspension of 5X 106 lymphocytes was centrifuged at 400g and 4 ℃ for 5min and the supernatant discarded.
(2) Flick the tube bottom to loosen the cell pellet. 1mL of Trizol reagent was added to lymphocytes, the mixture was blown and mixed by a gun, and the mixture was left at room temperature for 5min to lyse the cells sufficiently.
(3) 200 μ L of chloroform was added, and after shaking manually and vigorously for 15s, the mixture was allowed to stand at room temperature for 5 min.
(4) Centrifuge at 12000g for 15min at 4 ℃ and aspirate the upper aqueous phase and transfer to another centrifuge tube, approximately 450. mu.L.
(5) Adding 500 μ L isopropanol, mixing, and standing at room temperature for 10 min.
(6) Centrifuging at 12000g for 10min at 4 deg.C, discarding supernatant, and precipitating RNA at the bottom of the tube.
(7) 1mL of 75% ethanol (prepared in DEPC water) was added and the tubes were gently shaken to suspend the RNA pellet in whole pieces.
(8) centrifuging at 4 deg.C for 5min at 7500g, discarding supernatant, standing at room temperature for 5-8min, and volatilizing alcohol to prevent over drying.
(9) dissolving the RNA precipitate with 30 μ L DEPC/non-enzyme water, packaging, labeling, and storing in refrigerator at-80 deg.C.
(10) taking 2 mu L of RNA, detecting the concentration and OD value by using a micro ultraviolet spectrophotometer, taking 2 mu L of RNA electrophoresis detection strip, using 2% gel and DL2000 Marker, and simultaneously carrying out reverse transcription experiment immediately.
The detection result is that the total amount of RNA is more than 5 mug, the volume is more than 20 muL, the OD260/280 ratio is more than 1.8, and simultaneously, 2% agarose electrophoresis detection shows that three bands are 28sRNA, 18sRNA and 5sRNA, wherein 28S is 1-2 times of the brightness of 18S bands, and 5S bands are the lightest (shown in figure 1).
Example 2 reverse transcription and PCR amplification
1. RT-PCR (reverse transcription and first round PCR)
The extracted RNA was immediately reverse transcribed using QIAGEN onestrep RT-PCR kit (cat # 210212) and then subjected to the first round of amplification successively in the same system. The reverse transcription and PCR reaction systems are shown in Table 1, and the reaction procedures are shown in Table 2.
TABLE 1 reaction System
TABLE 2 reaction procedure
Wherein, TRTmix is a reverse transcription primer and also serves as a PCR reaction 3' end primer, the name and the sequence are shown in Table 3, each primer is mixed at an equal molar ratio concentration and then diluted to a total concentration of 20 mu M, or each primer is firstly prepared into a 20 mu M solution and then mixed according to the equal volume.
TABLE 3 reverse transcription primers
The V beta mix primers are 5' end multiplex PCR primers, the names and sequences of the primers are shown in table 4, the primers are mixed at equal molar ratio concentration and then diluted to total concentration of 20 mu M, or the primers are firstly prepared into 20 mu M solution and then mixed according to equal volume.
TABLE 4PCR primers
2. Second round PCR
The TCR region was specifically amplified using the Coco Taq enzyme (cat. TK01015) and the reaction system and conditions are shown in tables 5 and 6.
TABLE 5PCR reaction System
TABLE 6PCR reaction procedure
wherein, the V beta mix primer is a 5' end multiplex primer, is the same as the V beta mix of the RT-PCR reaction, and has the concentration of 20 mu M.
The 3 'hTCRCbBCX primer (20. mu.M) was selected from one of the primers in Table 7, the primers in Table 7 contained different barcocode sequences, one hTCRCbBCX primer was selected for one sample, one sample corresponded to only one barcode (capital letters), and the 3' primers of 6 samples in this example were SEQ ID NO:41-SEQ ID NO:46, respectively.
TABLE 7 specific sequences
| hTCRCbBCX | Sequence 5 '-3' | Numbering |
| hTCRCbBC1 | gagTTACTCGCgcacagcgacctcgggtgggaac | SEQ ID NO:41 |
| hTCRCbBC2 | gagTCGTTAGCgcacagcgacctcgggtgggaac | SEQ ID NO:42 |
| hTCRCbBC3 | gagTACCGAGCgcacagcgacctcgggtgggaac | SEQ ID NO:43 |
| hTCRCbBC4 | gagTGTTCTCCgcacagcgacctcgggtgggaac | SEQ ID NO:44 |
| hTCRCbBC5 | gagTTCGCACCgcacagcgacctcgggtgggaac | SEQ ID NO:45 |
| hTCRCbBC6 | gagTTGCGTACgcacagcgacctcgggtgggaac | SEQ ID NO:46 |
| hTCRCbBC7 | gagTCTACGACgcacagcgacctcgggtgggaac | SEQ ID NO:47 |
| hTCRCbBC8 | gagTGACAGACgcacagcgacctcgggtgggaac | SEQ ID NO:48 |
| hTCRCbBC9 | gagTAGAACACgcacagcgacctcgggtgggaac | SEQ ID NO:49 |
| hTCRCbBC10 | gagTCATCCTAgcacagcgacctcgggtgggaac | SEQ ID NO:50 |
3. Electrophoretic detection
The amplification effect of step 2 is detected by electrophoresis, and the target band is about 350bp, which is shown in figure 2.
4. Magnetic bead purification
And purifying one sample after 1-2 tubes of second round PCR. To the PCR product, 1.4 volumes of DNA fragments were added to sort and purify magnetic beads (BMSX BioMark BMSX), and eluted with 30. mu.L of water. The concentration of the purified product was measured using a microspectrophotometer.
5. 6 different barcode samples (up to 10, corresponding to 10 hTCRCbBCX primers) were mixed at equal concentrations, pooled, sequenced and subsequently analyzed.
Example 3 construction of libraries, sequencing and data analysis to obtain an Immunogram
1. Library construction and high throughput sequencing
(1) building warehouse
The procedure was followed exactly as in the manual, with sequencing adapters and quantification.
The Library is constructed according to the Kit instruction, and comprises an Ion Plus Fragment Library Kit (cat No. 4471252), an Ion Xpress Barcode Adaptors 1-16Kit (cat No. 4471250) and an Agencourt AMPure XP (cat No. A63881), and the instrument comprises a PCR instrument, a Qubit quantifier and a nucleic acid quantifying reagent.
(2) Template preparation and sequencing on machine
The Ion chef-automatic template preparation instrument can automatically perform experiments after reagents and consumables are installed, and the work of adding ISP magnetic beads, enrichment and loading chips to the library is completed.
Ion S5-sequencer to complete sequencing work and primary data processing.
Reagents used include Ion 520/530ExT-Chef-4rxns &4 initNEW-For 600bp (cat # A30670), Ion 530 Chip Kit (cat # A27764).
2. Analyzing data to obtain immune map
After high throughput sequencing was completed, data analysis was performed using FQ files, the sequences were subjected to IgBLAST, from which they were retrieved for use in D50Calculating the functional CDR3 amino acid sequences of different types, and counting the corresponding number, wherein the number is sorted from high to low, and the file name is the number of a subject, namely a production CDR3 count. The method comprises the following specific steps:
(1) using web free platform analytics, https:// usegalaxy. And carrying out reverse processing on the original data FQ file.
(2) And combining the forward and reverse data and then separating the barcode to obtain a single sample FQ file.
(3) And performing FastQC quality inspection on the single sample FQ file, and downloading the result.
(4) The reverse processing is again performed on the single sample FQ file.
(5) And carrying out format conversion on the FQ file subjected to the reverse processing to obtain a Fasta file.
(6) VDJ data comparison (free platform Igblast comparison is recommended, and https:// www.ncbi.nlm.nih.gov/Igblast /) is carried out on a single sample Fasta file to obtain a comparison file.
(7) And (4) capturing comparison data of the functional sequences by comparing files, and storing the comparison data separately under the file name production.
(8) And further capturing different types of CDR3 amino acid sequences from the comparison data of the functional sequences, and counting the corresponding number, wherein the number is sorted from high to low and is separately stored, and the file name is productive _ CDR3_ count.
production _ CDR3_ count file for D50The specific calculation method is as follows:
Sequencing the obtained TCR variable region sequence information, wherein the total number of CDR3 sequences of all functional gene variable regions is represented as N, the number of types of CDR3 of different sequences (clones) is represented as C, the copy number of each clone is represented as N1, N2 … … NC and is sorted from the copy number to the copy number (N1 is more than or equal to N2 is more than or equal to … … NC-1 is more than or equal to NC), the number of types of clones with the largest copy number and occupying 50 percent of the total number of functional CDR3 sequences is represented as H, and the ratio of H to C is defined as D50。
Example 4 evaluation of the level of Immunity of a subject
Data analysis of 6 subjects and D50The calculation results are as follows:
TCR sequencing data were processed, wherein QC results are shown in Table 8, D50The relevant data statistics are shown in table 9.
TABLE 8 test results of QC of subjects
TABLE 9D50detecting data
3. According to D50Data, prediction of the efficacy of a subject using an immunodetection point modulating class of drugs:
(1) If it isD50<0.01, indicating that the immunity of the testee is low, and the remarkable curative effect is difficult to obtain by using the immunodetection point regulating medicines;
(2) If D is 0.01. ltoreq.D50Less than or equal to 0.015 shows that the immunity of the testee is poor, and the use of the immunodetection point regulation medicines can possibly achieve certain effect;
(3) If 0.015<D50The method shows that the immunity of the testee is relatively good, and the immune checkpoint point regulating medicines are used to obtain a significant effect.
Wherein, the definition of the curative effect of the immunodetection point regulation type medicine is as follows:
(1) Complete remission (Complete Release, CR): all target lesions disappeared, no new ones appeared, and tumor markers were normal for at least 4 weeks.
(2) Partial remission (Complete Release, CR): the sum of the maximum diameters of the target lesions is reduced by more than or equal to 30 percent and is maintained for at least 4 weeks.
(3) Stable Disease (SD): the sum of the maximum diameters of the target lesions reduces the underreached PR or increases the underreached PD.
(4) disease Progression (PD): the sum of the maximum diameters of the target lesions is at least increased by more than or equal to 20 percent, or new lesions appear.
EXAMPLE 5 predictive assessment of the efficacy of immunodetection Point-modulated drugs
Immunopogram assays were performed on 31 lymphoma subjects according to the method described in example 1, all subjects corresponding to D50The values are shown in Table 10:
TABLE 10 Immunopogram detection assay for lymphoma Subjects
Ideally, each CDR3 clone has only one functional copy, D500.5; when the immunity of the subject is reduced or the subject is suffering from tumor and immunityIn case of diseases such as deficiency, D50The value drops sharply. Thus, subject health status and immune diversity (D)50) Has significant correlation.
Classifying according to the curative effect of the subject after receiving the immunodetection point regulation type drug treatment, and comparing with the subject D50The numerical values were analyzed. Complete Remission (CR) of patients, D50the mean value was 0.03 and the median value was 0.018, all significantly higher than that of the other patients (Non-CR) (see table 11, fig. 3).
TABLE 11 immunodetection Point modulation drug efficacy and D50Relationship of numerical values
Then according to D50subjecting the subject to classification analysis when D50<Complete remission (6.7%) was obtained in only 1 of 0.01, 15 subjects after immunodetection point-modulating drugs; when D is more than or equal to 0.0150At ≦ 0.015, complete remission (57.1%) was obtained in 4 of 7 subjects; when 0.015<D50Complete remission (77.8%) was obtained in 7 out of 9 subjects. Thus, subject immune diversity (D)50) Can be used to predict the prognosis using immunodetection point-modulating drugs (Table 12, FIG. 4).
TABLE 12D50Relationship between numerical value and curative effect of immunodetection point regulation type medicine
thus, we define the criteria for assessing the prognosis of a subject using an immunodetection point-modulating class of drugs by immunoprography analysis as:
(1) When D is present50<When 0.01 hour, the immunity of the patient is low, and the remarkable curative effect is difficult to obtain by using the immunodetection point regulating medicines;
(2) When D is more than or equal to 0.0150when the content is less than or equal to 0.015, the immunity of the patient is poor, and a certain effect can be obtained by using the immunodetection point regulation type medicine;
(3) When 0.015<D50The preparation method shows that the immunity of the patient is relatively good, and the application of the immunodetection point regulating medicine has a great possibility of obtaining a remarkable effect.
Thus, in summary, an index D indicating the diversity of CDR3 can be determined50As an indicator for assessing the prognosis of a subject using an immunodetection point modulating drug.
Sequence listing
<110> Chengdu Yianbo Biotech Ltd
<120> RT-PCR kit for predicting curative effect of immunodetection point regulation type medicines and prediction method thereof
<160> 50
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgggagat 8
<210> 2
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cttttggg 8
<210> 3
<211> 8
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ccagtgtg 8
<210> 4
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ctctgcttct gatggctcaa acacagc 27
<210> 5
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggtgctgtcg tctctcaaca tccgag 26
<210> 6
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
agtgctgtca tctctcaaaa gccaagcag 29
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tctcagacta ttcatcaatg gccagcgacc 30
<210> 8
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gatgctgtag ttacacaatt cccaagacac ag 32
<210> 9
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gaagctgaca tctaccagac cccaagatac c 31
<210> 10
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gatgctgatg ttacccagac cccaaggaat a 31
<210> 11
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gaagcccaag tgacccagaa cccaagatac 30
<210> 12
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gatgtgaaag taacccagag ctcgagatat ctagtc 36
<210> 13
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gatggtggaa tcactcagtc cccaaagtac c 31
<210> 14
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gatgctgaaa tcacccagag cccaag 26
<210> 15
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gatgctgaaa tcacccagag cccaag 26
<210> 16
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gatgctgaaa tcacccagag cccaag 26
<210> 17
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gatgctgaaa tcacccagag cccaag 26
<210> 18
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gatgccatgg tcatccagaa cccaagatac 30
<210> 19
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gatgccatgg tcatccagaa cccaagatac 30
<210> 20
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cccagacacc aaaatacctg gtcacagc 28
<210> 21
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cacctggtca tgggaatgac aaataagaag tc 32
<210> 22
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gctgctggag tcatccagtc ccc 23
<210> 23
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gattctggag tcacacaaac cccaaagcac 30
<210> 24
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
agatatctga tcaaaacgag aggacagca 29
<210> 25
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
acacacctga tcaaaacgag aggacagca 29
<210> 26
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
aatgccggcg tcatgcagaa cccaaga 27
<210> 27
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ggtgaagaag tcgcccagac tccaaaac 28
<210> 28
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gacaccaagg tcacccagag acctag 26
<210> 29
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gagcctggag tcagccagac cc 22
<210> 30
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
cccaggcaca aagtgacaga gatgggacaa 30
<210> 31
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ccccggcacg aggtgacaga gatgggacaa 30
<210> 32
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gaagctggag ttactcagtt ccccagc 27
<210> 33
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gaacctgaag tcacccagac tcccagc 27
<210> 34
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gaagctggag tggttcagtc tcccag 26
<210> 35
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gaagctggag tggttcagtc tcccag 26
<210> 36
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ccccagacac aagatcacaa agaggggac 29
<210> 37
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
ctcccaggta caaagtcaca aagaggggac 30
<210> 38
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
cccctaggta caaagtcgca aagagaggac 30
<210> 39
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
ccctgagaca caaggtagca aagaagggaa a 31
<210> 40
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
cccccagtaa caaggtcaca gagaagggaa a 31
<210> 41
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
gagttactcg cgcacagcga cctcgggtgg gaac 34
<210> 42
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
gagtcgttag cgcacagcga cctcgggtgg gaac 34
<210> 43
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gagtaccgag cgcacagcga cctcgggtgg gaac 34
<210> 44
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gagtgttctc cgcacagcga cctcgggtgg gaac 34
<210> 45
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
gagttcgcac cgcacagcga cctcgggtgg gaac 34
<210> 46
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
gagttgcgta cgcacagcga cctcgggtgg gaac 34
<210> 47
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
gagtctacga cgcacagcga cctcgggtgg gaac 34
<210> 48
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
gagtgacaga cgcacagcga cctcgggtgg gaac 34
<210> 49
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gagtagaaca cgcacagcga cctcgggtgg gaac 34
<210> 50
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
gagtcatcct agcacagcga cctcgggtgg gaac 34
Claims (6)
1. An RT-PCR kit for predicting the curative effect of immunodetection point regulation medicines is characterized by comprising a primer for RT-PCR amplification and auxiliary components thereof; the primers comprise a primary RT-PCR amplification primer and a specific primer for performing secondary PCR amplification on a TCR region of a primary amplification product;
The first-round RT-PCR amplification primer is shown as SEQ ID NO. 1-40; the second round PCR amplification specific primer is shown as SEQ ID NO. 41-50.
2. The RT-PCR kit for predicting the curative effect of immunodetection point-regulated drugs according to claim 1, wherein the auxiliary components comprise RT-PCR buffer solution, MgCl2、dNTP、ddH2O, and an enzyme.
3. a method for predicting the efficacy of an immunodetection point modulating drug using a kit according to claim 1 or 2, comprising the steps of:
(1) Extracting RNA of a sample to be detected;
(2) Carrying out reverse transcription on an RNA template of a sample to be detected, then carrying out first-round RT-PCR amplification on cDNA prepared by the reverse transcription, and collecting a purified amplification product;
(3) Performing second round of PCR amplification on the amplification product obtained in the step (2) again to amplify the TCR variable region to obtain a variable region sequence;
(4) Calculating D based on the variable region sequence obtained in step (3)50The value is obtained.
4. The method of claim 3, wherein D is performed in step (4)50The values are calculated as follows: sequencing to obtain TCR variable region sequence information, comparing to determine the total number of variable region sequences as N, the number of types of sequences different from variable region genes as C, the copy number of each sequence is N1 and N2 … … NC, sequencing the sequences according to the copy number from at least N1 and N2 … … NC, the number of clone types with the most copy number and occupying 50 percent of the total number of functional variable region sequences is H, and the ratio of H to C is D50。
5. The method according to claim 3, wherein the TRTmix primers in the reaction system of the first RT-PCR amplification in the step (2) are mixed by the primers shown in SEQ ID NO: 1-4 at an equimolar concentration and then diluted to 20 μ M, or the primers in the primers shown in SEQ ID NO: 1-4 are respectively adjusted to 20 μ M in concentration and then mixed at an equal volume;
The V beta mix primers are mixed by the primers shown by SEQ ID NO. 5-40 in an equimolar concentration ratio, and then diluted to 20 mu M, or the concentration of each primer in the primers shown by SEQ ID NO. 5-40 is respectively adjusted to 20 mu M, and then the primers are mixed in an equimolar volume.
6. The method of claim 3, wherein the sequence uniqueness of the TCR variable region is determined by the CDR3 sequence.
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Cited By (4)
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| CN111693702A (en) * | 2020-07-11 | 2020-09-22 | 成都益安博生物技术有限公司 | Peripheral blood TCR marker of melanoma and detection kit and application thereof |
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| CN113684258A (en) * | 2020-05-18 | 2021-11-23 | 上海赛比曼生物科技有限公司 | Kit and method for detecting mouse-derived TCR transgene copy number |
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Application publication date: 20191213 |
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| RJ01 | Rejection of invention patent application after publication |