CN112029833A - Rapid identification method of CTNNB1 gene mutation for tumor organoid culture condition selection - Google Patents
Rapid identification method of CTNNB1 gene mutation for tumor organoid culture condition selection Download PDFInfo
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Abstract
A rapid identification method of CTNNB1 gene mutation for selecting tumor organoid culture conditions belongs to the technical field of gene detection, and is characterized in that: the detection of single and multiple CTNNB1 gene mutations of tumor tissues; the method comprises the steps of autonomously setting and screening variant sites according to CTNNB1 mutation site information related to carcinogenesis, designing special primers and probes based on an ARMS-PCR method of a real-time fluorescence quantitative PCR instrument, and establishing the method through experimental verification of single detection or multiple simultaneous detections on plasmid DNA with different CTNNB1 gene mutations. The method can identify whether the tumor tissue contains CTNNB1 gene mutation within 4 hours, can accurately and efficiently determine the corresponding tumor organoid culture conditions, has the technical advantages of strong operability, high accuracy and high sensitivity, has stable detection result and obvious effect, and has remarkable advantages.
Description
Technical Field
The invention relates to a rapid identification method of CTNNB1 gene mutation for selecting tumor organoid culture conditions, belonging to the technical field of gene detection.
Background
As a cancer major country, China has high cancer incidence rate, and new cases of the year account for about one fourth of the total number of the whole world. The latest national cancer statistics data report released by the national cancer center shows: in 2015, about 392.9 ten thousand people had the malignant tumor, the incidence rate was 285.83/10 ten thousand, and about 233.8 ten thousand people died, which means that more than 1 ten thousand people were diagnosed with cancer on average per day, and 7.5 people were diagnosed with cancer per minute. In more than ten years, the number of cancer diseases and deaths in China has continuously increased, the incidence of malignant tumors is increased by about 3.9% every year, and the mortality is increased by about 2.5% every year. At present, with the continuous and deep research of targeted drugs, a plurality of drugs targeting specific oncogenes continuously enter the clinic, and the good news is brought to the majority of patients. However, since the nature of cancer is a disease affected by multigenic mutations, patient tumors evolve continuously as the disease progresses, resulting in inter-patient tumor heterogeneity and intra-tumor heterogeneity. Cancer heterogeneity adds uncertainty to targeted precision therapy and is also one of the major causes of drug resistance in cancer drug therapy. Therefore, the development of precise medical treatment aiming at cancer heterogeneity has important practical significance, and the rise of tumor organoid technology provides a brand-new more effective application platform for the development of the precise medical treatment aiming at the cancer heterogeneity.
Organoids are three-dimensional microstructures created by 3D culture of cells or tissues with adult stem cell potential in vitro. Organoid culture based on patient tumor tissue is referred to as tumor organoid culture. The tumor organoids have the capability of autonomous growth and updating, and can retain the characteristics of tumor tissues and certain genetic stability, so compared with the traditional two-dimensional cultured tumor cell lines, the tumor organoids have higher clinical relevance and individual diversity, and are the in vitro tumor disease models which are closest to the clinical conditions at present. In technical application, the tumor organoid disease model can be used for guiding the selection of the drug administration of a patient in the precise medical treatment of cancer, and meanwhile, in the research and development of anti-cancer drugs, based on the establishment of a specimen library which maintains the heterogeneity of the cancer of the patient, the tumor organoid model can more effectively evaluate the drug effect of a lead compound in preclinical or clinical tests on specific types of cancer. Tumor organoids have become the best disease model that not only simulates the "environment" of tumor growth but also has patient specificity (superior to experimental animal models) in tumor basic research, and have brought dawn for the dilemma of "there is no target point in sequencing, there is no drug in the target point, there is no curative effect in the drug" in clinical research and treatment.
Just because tumor organoids can retain the properties of the original tumor, through drug testing of tumor organoids, treatment regimens that take into account cancer heterogeneity can be selected for patients of tumor origin, thereby facilitating further research and development of accurate cancer treatment. However, the culture technique of tumor organoids faces some unknowns and challenges. One of the challenges is that normal tissues are often mixed in tumor tissues, if a selected culture medium is incorrect, along with subculture, organoids derived from the normal tissues can gradually replace tumor organoids, so that a cultured tumor organoid group gradually loses tumor characteristics, experimental data of non-patient actual tumor conditions are obtained through drug sensitivity test aiming at the tumor organoids, and misguidance is generated on clinical medication, so that the time for selecting accurate and effective drugs by patients is delayed.
Currently, a variety of approaches have been used to address the above challenges. Taking liver cancer tumor organoids as an example, researchers have adopted four culture methods for specific screening of tumor organoids based on the difference between the tumor organoids in terms of physical properties, cellular properties and tumor-specific gene mutation from normal organoids, but these methods all have obvious limiting factors such as complicated operation or high requirements for laboratories. The first method is a manual operation treatment in the culture process, namely, a tool is used under a microscope to remove the organoids without the characteristics of cancer organoids and with normal phenotype, and the method has low feasibility and high error rate; the second method is to utilize a flow cytometer to sort and extract cancer stem cells in tumor tissue cells, and utilize the screened high-purity specific tumor cells to carry out organoid culture, so that the method has high requirements on hardware, and simultaneously has the risk that the selected specific cancer cells cannot reflect the main tumor properties of patients; the third method is to adjust the medium composition and screen organoids showing specific cancer-associated mutant phenotypes based on known patient sequencing results, which has the problem that the patient sequencing results are often obtained later than the initial culture time of tumor organoids; the fourth method is to determine whether there is a mutation associated with cancer in a patient by sequencing a tumor organoid obtained by culture, and has problems of high cost and low efficiency.
In many cancer tissues, Wnt-related mutations play an important role. The Wnt signal pathway is closely related to physiological processes such as animal embryonic development, organ formation, tissue regeneration and the like, the activation mutation of the Wnt signal pathway can directly cause the formation of tumors, and the beta-catenin coded by the CTNNB1 gene is a key factor of the signal pathway. The CTNNB1 gene mutation belongs to one of high-frequency mutations in cancers, has high pathogenicity to various cancers such as liver cancer, colorectal cancer, breast cancer and the like, and has wide and important cancer treatment guiding function aiming at the screening of CTNNB1 gene mutation. Therefore, for the obvious problems faced by the current tumor organoid specific screening culture method, how to quickly screen the tumor tissues of patients which can use organoid culture conditions related to CTNNB1 gene mutation before the culture is started becomes a technical problem to be solved urgently. Therefore, it is necessary to develop a rapid identification method of CTNNB1 gene mutation for tumor organoid culture condition selection.
Disclosure of Invention
In order to solve the technical problem of how to quickly screen out patient tumor tissues which can use organoid culture conditions related to CTNNB1 gene mutation before culture initiation, the invention provides a method for quickly identifying CTNNB1 gene mutation for selection of tumor organoid culture conditions. The identification method adopts an ARMS-PCR technology of a real-time fluorescent quantitative PCR instrument, designs a special Mutation primer, an ARMS-PCR amplification primer and a probe aiming at a group of Mutation sites of CTNNB1 genes related to cancer occurrence, and establishes a rapid screening method aiming at CTNNB1 gene Mutation. Through experimental verification of single detection or simultaneous detection of plasmid DNA with different CTNNB1 gene mutations, the identification method has strong specificity and high sensitivity, can quickly detect whether CTNNB1 gene mutations exist in tumor tissues at one time (within 4 hours), accurately and efficiently determines corresponding tumor organoid culture conditions, thereby improving the culture accuracy and reducing meaningless resource waste, effectively shortening the period and cost of related detection, more accurately predicting the drug effect, selecting a treatment scheme, realizing quick selection of the culture method of the tumor organoids with CTNNB1 gene mutations, and better serving for accurate medical treatment.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the invention relates to a method for rapidly identifying CTNNB1 gene mutation for selecting tumor organoid culture conditions, which detects single CTNNB1 gene mutation of tumor tissues and simultaneously detects validity verification of a plurality of CTNNB1 gene mutations of the tumor tissues, and comprises the following experimental steps:
(1) screening out functional mutation sites of the tumor CTNNB1 gene reaching certain frequency;
(2) designing ARMS-PCR amplification primers and probes of functional mutation sites reaching certain frequency in the tumor CTNNB1 gene in the step (1);
(3) selecting a DNA fragment capable of containing the functional mutation site in the step (1) and an ARMS-PCR amplification primer thereof, and designing the PCR amplification primer;
(4) carrying out PCR amplification on the PCR amplification primer in the step (3) by taking normal human genome DNA as a template;
(5) designing a TA cloning primer for the DNA fragment obtained in the step (4), carrying out TA cloning, and carrying out transformation and amplification;
(6) designing a Mutation primer of the functional Mutation site reaching a certain frequency in the tumor CTNNB1 gene in the step (1);
(7) carrying out PCR amplification on the Mutation primer in the step (6) by taking the plasmid DNA in the step (5) as a template, and carrying out conversion amplification;
(8) selecting a proper amount of tissues of a sample to be detected, and extracting genome DNA of the sample to be detected;
(9) setting the plasmid DNA in the step (7) as a positive control, and carrying out PCR amplification on the ARMS-PCR amplification primer in the step (2) by taking the genome DNA of the sample to be detected in the step (8) as a template;
(10) and analyzing the detection result by using an instrument and outputting the result, and determining that the sample has mutation by observing that a curve and a Cp value appear in the result output by the real-time fluorescence quantitative PCR instrument and a negative control and a blank control have no curve and Cp value.
The functional mutation sites of the tumor CTNNB1 gene reaching a certain frequency are as follows: CTNNB1_ p.d32n, CTNNB1_ p.d32g, CTNNB1_ p.s33p, CTNNB1_ p.s33c, CTNNB1_ p.s33f, CTNNB1_ p.s45p, CTNNB1_ p.s 45f. In the names of the mutation sites, CTNNB1 is a gene name, and the encoded protein beta-catenin is a key factor in the Wnt signal pathway. CTNNB1 is followed by point mutation, for example, p.d32n is a point mutation, and the corresponding amino acid (position 32) is mutated from aspartic acid (D) to asparagine (N); d32g refers to the mutation position, the corresponding amino acid (position 32) is mutated from aspartic acid (D) to glycine (G); s33p refers to the mutation site, the corresponding amino acid (position 33) is mutated from serine (S) to proline (P); s33c refers to the mutation position, the corresponding amino acid (position 33) is mutated from serine (S) to cysteine (C); s33f refers to the mutation site, the corresponding amino acid (position 33) is mutated from serine (S) to phenylalanine (F); s45p refers to the mutation position, the corresponding amino acid (position 45) was mutated from serine (S) to proline (P); s45f refers to the mutation position, and the corresponding amino acid (position 45) was mutated from serine (S) to phenylalanine (F).
The sequence of the TA cloning primer of the functional mutation site of the tumor CTNNB1 gene reaching a certain frequency is as follows:
upstream 5'-gttaggtggttccctaagggattagg-3'
Downstream 5'-tcaacactcactatccacagttcagc-3'
The TA Cloning Kit (origin TA Cloning Kit) is a method of ligating a PCR fragment with a vector DNA having a 3' -T overhang. This refers to the primers required to obtain PCR fragments for TA cloning.
The tumor CTNNB1 gene reaches 7 functional Mutation sites of a certain frequency of Mutation primers (the Mutation primers are primers required by a point Mutation PCR experiment by taking non-Mutation plasmid DNA obtained by TA cloning as a template, and the sequences of the obtained mutant plasmid DNA, ARMS-PCR primers and probes are as follows:
CTNNB1_p.D32N
mutation primer: upstream 5'-gcaacagtcttacctgaactctggaatcca-3'
Downstream 5'-tcaggtaagactgttgctgccagtgac-3'
ARMS-PCR primers: upstream 5'-ggcagcaacagtcttaccgga-3'
Downstream 5'-tgcattctgactttcagtaaggcaa-3'
And (3) probe: 5'-tctggtgccactaccacagctcc-3'
CTNNB1_p.D32G
Mutation primer: upstream 5'-caacagtcttacctgggctctggaatccat-3'
Downstream 5'-cccaggtaagactgttgctgccagtga-3'
ARMS-PCR primers: upstream 5'-ggcagcaacagtcttaccttgg-3'
Downstream 5'-aactgcattctgactttcagtaaggc-3'
And (3) probe: 5'-tctggtgccactaccacagctcc-3'
CTNNB1_p.S33P
Mutation primer: upstream 5'-cagtcttacctggaccctggaatccattctgg-3'
Downstream 5'-ggtccaggtaagactgttgctgccag-3'
ARMS-PCR primers: upstream 5'-cagcaacagtcttacctggccc-3'
Downstream 5'-aaggactgagaaaatccctgttccc-3'
And (3) probe: 5'-tctggtgccactaccacagctcc-3'
CTNNB1_p.S33C
Mutation primer: upstream 5'-agtcttacctggactgtggaatccattctgg-3'
Downstream 5'-cagtccaggtaagactgttgctgcca-3'
ARMS-PCR primers: upstream 5'-gcagcaacagtcttacctggagtg-3'
Downstream 5'-gcattctgactttcagtaaggcaatg-3'
And (3) probe: 5'-tctggtgccactaccacagctcc-3'
CTNNB1_p.S33F
Mutation primer: upstream 5'-agtcttacctggactttggaatccattctggt-3'
Downstream 5'-aagtccaggtaagactgttgctgcca-3'
ARMS-PCR primers: upstream 5 '-tggcagcaacagatcttacccrggaatt-3'
Downstream 5'-tgcattctgactttcagtaaggcaatga-3'
And (3) probe: 5'-tctggtgccactaccacagctcc-3'
CTNNB1_p.S45P
Mutation primer: upstream 5'-actaccacagctcctcctctgagtggtaaaggc-3'
Downstream 5'-gaggagctgtggtagtggcaccagaa-3'
ARMS-PCR primers: upstream 5'-tggtgccactaccacagctcatc-3'
Downstream 5'-tgcattctgactttcagtaaggcaatg-3'
And (3) probe: 5'-cctcccaagtcctgtatgagtggga-3'
CTNNB1_p.S45F
Mutation primer: upstream 5'-actaccacagctccttttctgagtggtaaaggc-3'
Downstream 5'-aaaggagctgtggtagtggcaccagaa-3'
ARMS-PCR primers: upstream 5'-gccactaccacagctccgtt-3'
Downstream 5'-actgcattctgactttcagtaaggc-3'
And (3) probe: 5'-cctcccaagtcctgtatgagtggga-3'
And (4) conclusion: the rapid screening method based on CTNNB1 gene mutation provided by the invention is used for detecting 7 common mutation sites of a CTNNB1 gene in a Wnt signal channel and simultaneously detecting a plurality of CTNNB1 gene mutations, the verification results are positive, and a result curve graph and a Cp value of each mutation site can be obtained by using a real-time fluorescence quantitative PCR instrument. No gene mutation was detected in both the negative control and the blank control, indicating that the present invention is feasible for detecting single CTNNB1 gene mutation in tumor tissues and for simultaneously detecting multiple CTNNB1 gene mutations in tumor tissues.
The invention has the beneficial effects that: (1) the rapid identification method of CTNNB1 gene mutation for selecting tumor organoid culture conditions selects cancer hot spot mutation sites which are obviously related to the risk of suffering from cancer, and the detected mutation sites are more advanced and independent. The site selection of the present invention is therefore representative and independent. (2) The detection technology provided by the invention has obvious cost price advantage, higher sensitivity and larger flux in the aspect of early detection of tumor tissues, can realize detection of a plurality of single small samples, and can accurately guide organoid culture. (3) The rapid identification method of CTNNB1 gene mutation for selecting tumor organoid culture conditions provided by the invention can identify whether tumor tissues contain CTNNB1 gene mutation or not in a very short time (4 hours), and can accurately and efficiently determine the corresponding tumor organoid culture conditions. (4) The rapid identification method of CTNNB1 gene mutation for selecting tumor organoid culture conditions, provided by the invention, has the technical advantages of strong operability, high accuracy and high sensitivity, and has the advantages of stable detection result, obvious effect and remarkable advantage.
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The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a graph of the sequencing results of mutant plasmid DNA for a rapid identification method of CTNNB1 gene mutation for selection of tumor organoid culture conditions.
FIG. 2 is a diagram showing the electrophoresis result of PCR products prepared by using normal human genome DNA as a template and using the normal human genome DNA as a template in a rapid identification method of CTNNB1 gene mutation for tumor organoid culture condition selection. In the figure: DNA Ladder Marker, 2 PCR Product.
FIG. 3 is a diagram showing the electrophoresis results of mutant plasmid DNA obtained by using the plasmid DNA of step (2) as a template, preparing a Mutation reaction system, and treating with DMT enzyme, in a method for rapidly identifying a Mutation in CTNNB1 gene used for selection of tumor organoid culture conditions. In the figure: DNA Ladder Marker, 2.CTNNB1_ p.D32N, 3.CTNNB1_ p.D32G, 4.CTNNB1_ p.S33P, 5.CTNNB1_ p.S33C, 6.CTNNB1_ p.S33F, 7.CTNNB1_ p.S45P, 8.CTNNB1_ p.S45F.
FIG. 4 is a graph of the CTNNB1_ p.D32N results and Cp values of ARMS-PCR experimental results of a rapid identification method of mutations in the CTNNB1 gene for selection of tumor organoid culture conditions.
FIG. 5 is a graph of the CTNNB1_ p.D32G results and Cp values of ARMS-PCR experimental results of a rapid identification method of mutations in the CTNNB1 gene for selection of tumor organoid culture conditions.
Fig. 6 is a graph of CTNNB1_ p.s33p results and Cp values of the ARMS-PCR experimental results of a rapid identification method of CTNNB1 gene mutations for tumor organoid culture condition selection.
Fig. 7 is a graph of CTNNB1_ p.s33c results and Cp values of the ARMS-PCR experimental results of a rapid identification method of CTNNB1 gene mutations for tumor organoid culture condition selection.
Fig. 8 is a graph of CTNNB1_ p.s33f results and Cp values of the ARMS-PCR experimental results of a rapid identification method of CTNNB1 gene mutations for tumor organoid culture condition selection.
Fig. 9 is a graph of CTNNB1_ p.s45p results and Cp values of the ARMS-PCR experimental results of a rapid identification method of CTNNB1 gene mutations for tumor organoid culture condition selection.
FIG. 10 is a graph of the results of ARMS-PCR experiments with the result of CTNNB1_ p.S45F and Cp values for a method for rapid identification of mutations in CTNNB1 gene for selection of tumor organoid culture conditions
FIG. 11 is a graph of the results of multiple CTNNB1 mutations and Cp values from ARMS-PCR experiments for a method of rapid identification of mutations in the CTNNB1 gene for selection of tumor organoid culture conditions
Detailed Description
As shown in the figure, the related experimental processes of the rapid screening method based on CTNNB1 gene mutation are as follows:
the following examples are presented to illustrate certain embodiments of the invention in particular and should not be construed as limiting the scope of the invention. The present disclosure may be modified from materials, methods, and reaction conditions at the same time, and all such modifications are intended to be within the spirit and scope of the present invention. Specifically, the reagents used in the embodiments of the present invention are all commercially available products, and the databases used in the embodiments of the present invention are all public online databases.
The main reagents are as follows: pEASY-T1 Cloning kit, AmpliTaq
DNA Polymerase, LD, Vader-Trap high purity Plasmid miniprep Kit, Fast Mutagenesis System Kit, Hipure Plasmid Mini Kit, and,
Probe qPCR SuperMix(2×)。
The main apparatus is as follows: PCR instrument, LightCycler480 high-flux real-time fluorescence quantitative PCR instrument, super clean bench, liquid-transferring gun, gel electrophoresis instrument, water bath, incubator, shaking table, gel irradiation instrument, and high-speed centrifuge.
Example 1: the ARMS-PCR detection method provided by the invention is used for detecting single CTNNB1 gene mutation.
1. Study object
The 7 mutation sites were selected: CTNNB1_ p.D32N, CTNNB1_ p.D32G, CTNNB1_ p.S33P, CTNNB1_ p.S33C, CTNNB1_ p.S33F, CTNNB1_ p.S45P and CTNNB1_ p.S45F, and normal human genomic DNA was simultaneously set as a negative control and water (H.sub.p.D32N, CTNNB1_ p.D32G) was added to the whole culture of the human genome2O) as blank control.
2. Experimental procedure
(1) TA cloning primer primers, Mutation primers, ARMS-PCR amplification primers and probes for achieving a certain frequency of functional Mutation sites of the tumor CTNNB1 gene in the table 1, the table 2 and the table 3 are designed.
Table 1: TA cloning primer of normal human DNA fragment containing 7 mutation sites and ARMS-PCR amplification primer thereof
| Primer name | Primer sequence (5 '-3') |
| C-Primer-F | gttaggtggttccctaagggattagg |
| C-Primer-R | tcaacactcactatccacagttcagc |
Table 2: ARMS-PCR amplification primer and probe for functional mutation sites with certain frequency in tumor CTNNB1 gene
Table 3: mutation primer for achieving functional Mutation sites with certain frequency in tumor CTNNB1 gene
(2) Using normal human genome DNA as a template, and preparing a PCR reaction system according to the following table:
thermal cycling parameters of amplification:
| Step | Temperature(℃) | |
| 1 | 95 | 10：00 |
| 2 | 95 | 00：15 |
| 3 | 55 | 00：15 |
| 4 | 72 | 00：50 |
| 5 | Go to Step 2 | |
| 6 | 72 | 7：00 |
the electrophoresis results of the PCR products are shown in FIG. 2.
TA cloning reaction System:
| Component | Volume(μl) |
| |
4 |
| pEASY- |
1 |
| |
5 |
after cloning was successful, transformation and sequencing were performed.
(3) Taking the plasmid DNA in the step (2) as a template, and respectively preparing a Mutation reaction system with 7 Mutation sites according to the following table:
thermal cycling parameters of amplification:
| Step | Temperature(℃) | |
| 1 | 94 | 5：00 |
| 2 | 94 | 00：20 |
| 3 | 55 | 00：20 |
| 4 | 72 | 2：20 |
| 5 | Go to Step 2 | |
| 6 | 72 | 10：00 |
(4) the PCR product was treated with 1. mu.l DMT enzyme at 37 ℃ for 1 hour, electrophoretically detected, transformed and sequenced.
The electrophoresis results of the mutant plasmid DNA are shown in FIG. 3.
The sequencing results of the mutant plasmid DNA are shown in FIG. 1.
(5) And (3) respectively preparing ARMS-PCR reaction systems with 7 mutation sites by taking the mutant plasmid DNA in the step (4) as a template according to the following table:
at the same time, negative control of normal human genome DNA and water blank control were set up according to the above table.
Negative Control：
Blank Control：
Thermal cycling parameters of amplification:
the result curve graph and the Cp value of the ARMS-PCR experiment result are shown in figures 4-10, the sample to be tested has a curve and a Cp value, and the negative control and the blank control have no curve and no Cp value, so that the sample is determined to have mutation.
3. Conclusion
The establishment method is used for detecting 7 common mutation sites in the CTNNB1 gene, the verification result shows that the result is positive, a result curve chart and a Cp value can be obtained by using a LightCycler480 high-flux real-time fluorescence quantitative PCR instrument, and no gene mutation is detected in a negative control and a blank control, which shows that the method has feasibility.
Example 2: the ARMS-PCR detection method can simultaneously detect a plurality of CTNNB1 gene mutations.
1. Study object
The following 7 mutation sites were selected: CTNNB1_ p.D32N, CTNNB1_ p.D32G, CTNNB1_ p.S33P, CTNNB1_ p.S33C, CTNNB1_ p.S33F, CTNNB1_ p.S45P and CTNNB1_ p.S45F, and normal human genomic DNA was simultaneously set as a negative control and water (H.sub.p.D32N, CTNNB1_ p.D32G) was added to the whole culture of the human genome2O) as blank control.
2. Experimental procedure
(1) ARMS-PCR amplification primers and probes of Table 2 in example 1 were used.
(2) Using the 7 mutant plasmid DNAs of example 1 as templates, ARMS-PCR reaction systems were prepared using the probes and ARMS-PCR amplification primers as follows:
negative controls for normal human genomic DNA and water blank controls were also set as per the above table:
Negative Control：
Blank Control：
thermal cycling parameters of amplification:
the result curve graph and the Cp value of the ARMS-PCR experimental result are shown in FIG. 11, the sample to be tested has a curve and a Cp value, and the negative control and the blank control have no curve and Cp value, so that the sample is determined to have mutation.
3. Conclusion
The verification result shows positive, and a result graph and Cp value of each mutation site can be obtained by using a LightCycler480 high-throughput real-time fluorescence quantitative PCR instrument. No gene mutation was detected in both the negative control and the blank control, indicating that the present invention is feasible to detect multiple CTNNB1 gene mutations in cancer tissues simultaneously.
The invention provides a group of Mutation sites of CTNNB1 gene in Wnt signal path related to cancer occurrence based on ARMS-PCR technology, and designs special Mutation primers, ARMS-PCR amplification primers and probes, and experiments prove that the culture method for rapidly screening cancer organs based on CTNNB1 gene Mutation has strong specificity and high sensitivity, can detect possible Mutation sites at one time, and is accurate and efficient.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. The above-described embodiments are preferred embodiments of the present invention, and it should be understood by those skilled in the art that the above-described embodiments and descriptions are only illustrative of the principles of the present invention, but not limiting the embodiments of the present invention, and it should be noted that various changes and modifications can be made by those skilled in the art without departing from the inventive concept of the present invention, and all such changes and modifications should be considered as equivalent to the protection scope of the present invention.
Claims (4)
1. A rapid identification method of CTNNB1 gene mutation for tumor organoid culture condition selection can detect single CTNNB1 gene mutation of tumor tissue and/or simultaneously detect multiple CTNNB1 gene mutations of liver cancer tissue; the CTNNB1 gene is a protooncogene, and the gene mutation and overexpression of the CTNNB1 gene are related to various tumorigenesis; a CTNNB1 gene Mutation rapid identification method for tumor organoid culture condition selection is provided, which is characterized in that the method steps of screening Mutation sites are automatically set according to CTNNB1 Mutation site information related to carcinogenesis, and a special Mutation primer, an ARMS-PCR amplification primer and a probe are designed based on an ARMS-PCR method of a real-time fluorescence quantitative PCR instrument, and are established through experimental verification of single detection or multiple simultaneous detection of plasmid DNA with different CTNNB1 gene mutations; the method is characterized in that: the validity verification of the method for rapidly identifying the CTNNB1 gene mutation for selecting the tumor organoid culture condition to detect the single CTNNB1 gene mutation of the tumor tissue and simultaneously detect a plurality of CTNNB1 gene mutations of the tumor tissue comprises the following experimental steps:
(1) screening out functional variation sites with certain frequency of tumor CTNNB1 gene mutation;
(2) designing ARMS-PCR amplification primers and probes of functional mutation sites with certain frequency of CTNNB1 gene mutation in the liver cancer Wnt pathway in the step (1);
(3) selecting a DNA fragment capable of containing the CTNNB1 gene functional mutation site in the step (1) and an ARMS-PCR amplification primer thereof, and designing a PCR amplification primer;
(4) carrying out PCR amplification on the PCR amplification primer in the step (3) by taking normal human genome DNA as a template;
(5) designing a TA cloning primer for the DNA fragment obtained in the step (4), carrying out TA cloning, and carrying out transformation and amplification;
(6) designing a Mutation primer of a functional Mutation site with a certain frequency of CTNNB1 gene Mutation in the liver cancer Wnt pathway in the step (1);
(7) carrying out PCR amplification on the Mutation primer in the step (6) by taking the plasmid DNA in the step (5) as a template, and carrying out conversion amplification;
(8) setting the plasmid DNA in the step (7) as a positive control, and carrying out PCR amplification on the ARMS-PCR amplification primer in the step (2) by taking the genome DNA of the sample to be detected as a template;
(9) analyzing the detection result by using real-time fluorescence quantitative PCR instrument software and outputting the result, and judging whether the sample is mutated or not by observing a result curve graph Amplification details output by the software; the functional variation sites reaching a certain frequency in the tumor CTNNB1 gene are as follows: CTNNB1_ p.d32n, CTNNB1_ p.d32g, CTNNB1_ p.s33p, CTNNB1_ p.s33c, CTNNB1_ p.s33f, CTNNB1_ p.s45p, CTNNB1_ p.s 45f; the sequences of the Mutation primer, ARMS-PCR primer and probe reaching a certain frequency of functional Mutation sites in the tumor CTNNB1 gene are shown as Seq ID No: 3, Seq ID No: 4, Seq ID No: 5, Seq ID No: 6, Seq ID No: 7, Seq ID No: 8, Seq ID No: 9, Seq ID No: 10, Seq ID No: 11, Seq ID No: 12, Seq ID No: 13, Seq ID No: 14, Seq ID No: 15, Seq ID No: 16, Seq ID No: 17, Seq ID No: 18, Seq ID No: 19, Seq ID No: 20, Seq ID No: 21, Seq ID No: 22, Seq ID No: 23, Seq ID No: 24, Seq ID No: 25, Seq ID No: 26, Seq ID No: 27, Seq ID No: 28, Seq ID No: 29, Seq ID No: 30, Seq ID No: 31, Seq ID No: shown at 32.
2. The method for rapidly identifying the CTNNB1 gene mutation used for the selection of tumor organoid culture conditions according to claim 1, wherein the CTNNB1 gene mutation is characterized in that: the sequences of the TA cloning primers of the functional variant sites reaching certain frequency in the tumor CTNNB1 gene are shown as Seq ID No: 1 and Seq ID No: 2, respectively.
3. The method for rapidly identifying the CTNNB1 gene mutation used for the selection of tumor organoid culture conditions according to claim 1, wherein the CTNNB1 gene mutation is characterized in that: the ARMS-PCR detection method is used for detecting the research object of a single CTNNB1 gene of a tumor tissue, and the following 7 mutation sites are selected: CTNNB1_ p.D32N, CTNNB1_ p.D32G, CTNNB1_ p.S33P, CTNNB1_ p.S33C, CTNNB1_ p.S33F, CTNNB1_ p.S45P and CTNNB1_ p.S45F, and normal human genomic DNA was simultaneously set as a negative control and water (H.sub.p.D32N, CTNNB1_ p.D32G) was added to the whole culture of the human genome2O) as blank control.
4. The method for rapidly identifying the CTNNB1 gene mutation used for the selection of tumor organoid culture conditions according to claim 1, wherein the CTNNB1 gene mutation is characterized in that: the ARMS-PCR detection method can be used for simultaneously detecting the research objects of a plurality of CTNNB1 genes of tumor tissues, and the following 7 mutation sites are selected: CTNNB1_ p.D32N, CTNNB1_ p.D32G, CTNNB1_ p.S33P, CTNNB1_ p.S33C, CTNNB1_ p.S33F, CTNNB1_ p.S45P and CTNNB1_ p.S45F, and normal human genomic DNA was simultaneously set as a negative control and water (H.sub.p.D32N, CTNNB1_ p.D32G) was added to the whole culture of the human genome2O) as blank control.
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