Colorectal cancer medication gene joint detection specific primer group, kit, method and application

Abstract

The invention provides a colorectal cancer drug gene joint detection specific primer group, a kit, a method and application, the colorectal cancer drug gene joint detection specific primer group comprises 270 primer sequence groups for detecting colorectal cancer drug genes, each primer sequence group comprises a forward primer and a reverse primer, each primer sequence group comprises a forward primer and a reverse primer, and the sequences of the forward primer and the reverse primer are shown as SEQ ID NO 1-SEQ ID NO 540. According to the detection method and the kit, the gene variation condition of the colorectal cancer patient can be comprehensively and accurately detected, and powerful support is provided for a doctor to formulate a personalized precise treatment scheme. Through a detection result, a doctor can select the most suitable medicine and treatment scheme according to gene characteristics of a patient, invalid treatment is avoided, the treatment effect is improved, and pain and medical cost of the patient are reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of high-throughput detection of colorectal tumors, and particularly to a method and kit for combined detection of genes for colorectal cancer medication. Background Art

[0002] Colorectal cancer is one of the common malignant tumors worldwide, seriously threatening human health. With the development of precision medicine, personalized treatment based on gene detection has become the key to improving the treatment effect of colorectal cancer.

[0003] Currently, there are various types of drugs for clinical treatment of colorectal cancer, including chemotherapy drugs, targeted therapy drugs, and immunotherapy drugs, etc. However, different patients have significant differences in their responses to drugs. This difference is largely determined by gene mutations in patients. For example, some patients carry specific gene mutations, resulting in drug resistance to certain chemotherapy drugs, making the treatment effect poor. At the same time, existing gene detection technologies have certain limitations in terms of detection range, accuracy, and detection efficiency, and it is difficult to provide a basis for clinical treatment comprehensively and accurately.

[0004] The Chinese patent literature discloses "A method for gene detection of lung cancer and colorectal cancer based on the NGS method", with the publication number CN 116042790A. This invention uses NGS technology to detect genes of multiple tumor-related mutation hotspots in plasma samples of colorectal cancer and lung cancer patients, determines genes or gene combinations with strong specificity and high sensitivity in plasma, and conducts screening, diagnosis, and efficacy monitoring of colorectal cancer and lung cancer through plasma DNA detection. However, the detection results of this invention have low accuracy and are not comprehensive, and cannot accurately screen drugs for the treatment of colorectal cancer.

[0005] Therefore, it is of great clinical significance to develop a detection method and kit that can comprehensively detect gene mutations related to colorectal cancer and accurately evaluate the sensitivity and drug resistance of patients to various drugs. Summary of the Invention

[0006] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a method and kit for combined detection of genes for colorectal cancer medication, so as to solve the problem that existing gene detection technologies for colorectal cancer medication have certain limitations in terms of detection range, accuracy, and detection efficiency.

[0007] To achieve the above purpose and other related purposes, the present invention provides a specific primer set for combined detection of genes for colorectal cancer medication, including the following 270 primer sequence groups for detecting genes for colorectal cancer medication. Each primer sequence group includes a forward primer and a reverse primer, and the sequences of the forward primer and the reverse primer in each primer sequence group are shown as SEQ ID NO 1 to SEQ ID NO 540 in Table 1: Table 1. The primer sequence groups each include the sequences of the forward primer and the reverse primer

[0008] Based on the sequence information of 270 target genes, the present invention designs specific probes containing the above specific primer groups by using professional bioinformatics software. The length of the probes is generally between 20 and 100 bp to ensure that they can specifically hybridize with the target gene regions. After the design is completed, the probes are synthesized by a synthesis company, and the quality of the probes is detected to ensure the accuracy and stability of the probes.

[0009] The present invention also provides an application of a specific primer group for the combined detection of colorectal cancer drug - related genes in the preparation of a kit for detecting colorectal cancer drug - related genes.

[0010] The present invention also provides a kit for the combined detection of colorectal cancer drug - related genes, including a primer premix corresponding to the above - mentioned specific primer group for the combined detection of colorectal cancer drug - related genes.

[0011] The kit for the combined detection of multiple genes in colorectal cancer of the present invention carefully includes the above - mentioned 270 genes related to colorectal cancer drug resistance. These genes comprehensively cover all the genes recommended by the guidelines and the hot genes in current clinical research, ensuring that the detection results can reflect the gene status of patients to the greatest extent and providing a reliable basis for precision treatment.

[0012] The present invention provides a method for the combined detection of colorectal cancer drug - related genes. Based on the above - mentioned kit for the combined detection of colorectal cancer drug - related genes, it includes the following steps: (1) Extract DNA from the tumor tissue sample of a colorectal cancer patient and detect the concentration and purity of the DNA; (2) Fragment the extracted DNA to obtain fragmented DNA; (3) Use DNA ligase to connect sequencing adapters to the fragmented DNA to construct a library, and detect the quality and fragment distribution of the library; (4) Use the VariantBaits liquid hybridization capture kit and operate according to the kit instructions. Mix the constructed library with the probes for hybridization reaction to form a hybridization complex, enrich the hybridization complex, and remove the unbound DNA fragments to achieve enrichment of the target gene region; the probes are a set of 270 primer sequences for detecting colorectal cancer drug genes in the colorectal cancer drug gene combined detection kit. (5) Perform PCR amplification on the enriched target gene region to obtain an amplification product. (6) Perform high-throughput sequencing on the amplified product, conduct quality control and preprocessing on the sequencing data, and remove the low-quality reads. (7) Analyze the data processed in step (6), identify the mutation sites in the genes, compare the detected mutations with the clinical database, evaluate the relationship between the mutations and drug sensitivity and drug resistance, and generate a detection report.

[0013] The detection range of the colorectal cancer drug gene combined detection method of the present invention covers the genes related to drug resistance and sensitivity of common clinical drugs. Among them, the chemotherapy drugs mainly include fluorouracil (5-FU), oxaliplatin, and irinotecan, and these drugs are commonly used in combination treatment regimens such as FOLFOX and FOLFIRI. In the FOLFOX regimen, fluorouracil and oxaliplatin are used in combination. However, due to gene mutations, the efficacy of these two drugs is poor in some patients. By detecting the relevant genes with this kit, the response of patients to this regimen can be predicted in advance, providing a reference for doctors to adjust the treatment plan. For targeted therapy drugs, including monoclonal antibodies against epidermal growth factor receptor (EGFR) and monoclonal antibodies against vascular endothelial growth factor (VEGF). For example, certain patients have specific mutations in the EGFR gene, and the use of anti-EGFR monoclonal antibodies may have better therapeutic effects, and this kit can accurately detect these mutations to help doctors select more appropriate targeted therapy drugs. In addition, for immunotherapy drugs, immune checkpoint inhibitors (such as PD-1 inhibitors), their efficacy is also closely related to the gene status of patients, and this kit can detect the relevant genes to evaluate the potential response of patients to immunotherapy.

[0014] Preferably, in step (1), the A260 / A280 ratio of the extracted DNA sample is 1.8 - 2.0.

[0015] Preferably, in step (2), the length of the fragmented DNA is 150 - 300 bp.

[0016] Preferably, in step (3), the ligation reaction system includes DNA fragments, sequencing adapters, ligase, and buffer.

[0017] Preferably, in step (3), the quality and fragment distribution of the library are detected by agarose gel electrophoresis.

[0018] Preferably, in step (4), during the hybridization process, the probe specifically binds to the target gene region to form a hybridization complex; the hybridization complex is enriched by magnetic beads, and the unbound DNA fragments are removed.

[0019] Preferably, in step (5), the amplification reaction system includes the captured DNA sample, PCR primers, DNA polymerase, dNTPs, and buffer.

[0020] As described above, the specific primer set, kit, method, and application for the combined detection of colorectal cancer drug - related genes of the present invention have the following beneficial effects: (1) Using the VariantBaits liquid - phase hybridization capture sequencing technology, it has the characteristics of high sensitivity, high specificity, and high accuracy; it can effectively enrich the target gene region, improve the reliability of detection, reduce false - positive and false - negative results, and provide a more reliable basis for clinical diagnosis and treatment. (2) The present invention not only provides a practical detection tool for clinical treatment but also lays a foundation for the scientific research of colorectal cancer. By accumulating and analyzing the gene data of a large number of patients, the pathogenesis of colorectal cancer and the molecular basis of drug response can be further understood in depth, promoting the research and development in the field of colorectal cancer treatment. (3) The detection scope covers genes related to chemotherapy, targeted, and immunotherapy drugs, meeting the current needs of the diversified treatment of colorectal cancer; whether choosing traditional chemotherapy regimens or emerging targeted and immunotherapy, valuable gene information can be obtained through this kit, providing clinicians with a wider range of treatment options and more precise medication guidance. (4) The detection method and kit of the present invention can comprehensively and accurately detect the gene mutation situation of colorectal cancer patients, providing strong support for doctors to formulate personalized and precise treatment plans. Through the detection results, doctors can select the most suitable drugs and treatment plans according to the gene characteristics of patients, avoid ineffective treatment, improve the treatment effect, and reduce the pain and medical costs of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It shows a flow chart of the method for the combined detection of precision drugs for colorectal cancer. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The following describes the embodiments of the present invention through specific specific examples. Those skilled in the art can easily understand the other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0023] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments, rather than limiting the protection scope of the present invention; in the specification and claims of the present invention, unless otherwise clearly indicated in the text, the singular forms "a", "an", and "the" include the plural forms.

[0024] When an embodiment gives a numerical range, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials of the prior art similar to or equivalent to the methods, devices, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0025] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt the conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in this technical field.

[0026] The embodiment of the present application provides a colorectal cancer drug - using gene combined detection kit, including a primer premix corresponding to a primer set specific for the combined detection of 270 colorectal cancer drug - using genes. The primer set specific for the combined detection of colorectal cancer drug - using genes is characterized in that it includes the following 270 primer sequence groups for detecting colorectal cancer drug - using genes. Each primer sequence group includes a forward primer and a reverse primer, and the sequences of the forward primer and the reverse primer in each primer sequence group are shown as SEQ ID NO 1 - SEQ ID NO 540 in Table 1: Table 1. Sequences of the forward primer and the reverse primer in each primer sequence group

[0027] As Figure 1 shown, the embodiment of the present application provides a combined detection method for colorectal cancer drug - related genes. Based on the above - mentioned combined detection kit for colorectal cancer drug - related genes, it includes the following steps: (1) Sample DNA extraction: Use a commercial DNA extraction kit and extract DNA from the tumor tissue sample of the patient according to the operation steps in the kit instruction manual. During the extraction process, strictly control the experimental conditions to ensure the purity and integrity of the DNA. Detect the concentration and purity of the DNA by a spectrophotometer. The A260 / A280 ratio should be between 1.8 - 2.0 to ensure the accuracy of subsequent experiments; (2) Specific probe design: Design specific probes according to the sequence information of the 270 target genes in Table 1 above using professional bioinformatics software. The probe length is generally between 20 - 100bp to ensure that it can specifically hybridize with the target gene region. After design, synthesize the probes through a synthesis company and detect the quality of the probes to ensure the accuracy and stability of the probes; (3) Library construction: Fragment the extracted DNA. The DNA can be fragmented to an appropriate size, generally between 150 - 300bp, by using an ultrasonic crusher or enzymatic digestion method. Then, use DNA ligase to ligate the sequencing adapter to the fragmented DNA to construct a library. The ligation reaction system includes DNA fragments, sequencing adapters, ligase, and corresponding buffers, and the ligation reaction is carried out at an appropriate temperature and reaction time. After the reaction, detect the quality and fragment distribution of the library by agarose gel electrophoresis; (4) Liquid - phase hybridization capture: Use the VariantBaits liquid - phase hybridization capture kit and operate according to the kit instruction manual. Mix the constructed library with the probes and carry out a hybridization reaction at a specific temperature and reaction time. During the hybridization process, the probes specifically bind to the target gene region to form a hybridization complex. Enrich the hybridization complex by magnetic beads and remove the unbound DNA fragments to achieve enrichment of the target gene region; (5) Post-capture amplification: PCR amplification is performed on the enriched target gene region. The amplification reaction system includes the post-capture DNA sample, PCR primers, DNA polymerase, dNTPs, and buffer, etc. Appropriate PCR primers are designed according to the characteristics of the target gene to ensure the specificity and efficiency of amplification. The amplification reaction conditions include steps such as pre-denaturation, denaturation, annealing, and extension. By optimizing the reaction conditions, a sufficient amount of amplified products can be obtained.

[0028] (6) Deep sequencing and data analysis: The amplified products are subjected to high-throughput sequencing. After sequencing is completed, quality control and preprocessing are performed on the sequencing data to remove low-quality reads. Then the data is analyzed to identify mutation sites in the gene. The detected mutations are compared with the clinical database to evaluate the relationship between mutations and drug sensitivity and drug resistance, and a detailed test report is generated to provide medication suggestions for clinicians.

[0029] The above embodiments merely illustrate the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A specific primer set for combined detection of colorectal cancer drug genes, characterized in that: The invention comprises the following 270 primer sequence sets for detecting drug genes for colorectal cancer, each primer sequence set comprises a forward primer and a reverse primer, and each primer sequence set comprises the sequences of the forward primer and the reverse primer as shown in SEQ ID NO 1 to SEQ ID NO 540:

2. Use of the specific primer set for combined detection of colorectal cancer drug genes as claimed in claim 1 in the preparation of a kit for detecting colorectal cancer drug genes.

3. A colorectal cancer drug gene combined detection kit, characterized in that: It comprises primer premixes respectively corresponding to the specific primer set for combined detection of colorectal cancer drug genes as described in claim 1.

4. A method for combined gene detection of colorectal cancer drugs, characterized in that: The colorectal cancer drug gene combined detection kit according to claim 3 comprises the following steps: (1) Extract DNA from tumor tissue samples of colorectal cancer patients and detect the concentration and purity of the DNA; (2) fragmenting the extracted DNA to obtain fragmented DNA; (3) Use DNA ligase to connect the sequencing adapter to the fragmented DNA, construct a library, and detect the quality and fragment distribution of the library; (4) mixing the constructed library with the probe for hybridization reaction to form a hybridization complex, enriching the hybridization complex, and removing unbound DNA fragments to achieve enrichment of the target gene region; the probe is a primer sequence set of 270 for detecting colorectal cancer drug genes in a colorectal cancer drug gene combined detection kit; (5) performing PCR amplification on the enriched target gene region to obtain an amplified product; (6) Perform high-throughput sequencing on the amplified products, perform quality control and preprocessing on the sequencing data, and remove low-quality reads; (7) Analyze the data processed in step (6), identify the mutation sites in the gene, compare the detected mutations with the clinical database, evaluate the relationship between the mutations and drug sensitivity and resistance, and generate a test report.

5. The method for combined gene detection of colorectal cancer drugs according to claim 4, characterized in that: In step (1), the A260 / A280 ratio of the extracted DNA sample is 1.8 to 2.

0.

6. The method for combined gene detection of colorectal cancer drugs according to claim 4, characterized in that: In step (2), the length of the fragmented DNA is 150 to 300 bp.

7. The method for combined gene detection of colorectal cancer drugs according to claim 4, characterized in that: In step (3), the ligation reaction system includes DNA fragments, sequencing adapters, ligase and buffer.

8. The method for combined gene detection of colorectal cancer drugs according to claim 4, characterized in that: In step (3), the quality and fragment distribution of the library are detected by agarose gel electrophoresis.

9. The method for combined gene detection of colorectal cancer drugs according to claim 4, characterized in that: In step (4), during the hybridization process, the probe specifically binds to the target gene region to form a hybridization complex; the hybridization complex is enriched by magnetic beads to remove unbound DNA fragments.

10. The method for combined gene detection of colorectal cancer drugs according to claim 4, characterized in that: In step (5), the amplification reaction system includes the captured DNA sample, PCR primers, DNA polymerase, dNTPs and buffer.

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