A marker detection reagent, kit and application for intestinal cancer gene mutation detection

Abstract

This invention, entitled "A Biomarker Detection Reagent, Kit, and Application for Colorectal Cancer Gene Mutation Detection," belongs to the field of biomedical technology. The technical problem to be solved is to provide a biomarker detection reagent capable of comprehensively detecting colorectal cancer gene mutations while ensuring high sensitivity and high specificity, thus addressing the difficulty in simultaneously achieving comprehensive site coverage, high sensitivity, high specificity, and high accuracy in existing technologies. The key technical solution is a biomarker detection reagent for colorectal cancer gene mutation detection, comprising a combination of amplification primers for detecting mutation sites in KRAS, NRAS, PIK3CA, and BRAF genes. The amplification primer combination includes primers for amplifying blocked mutation systems and ordinary primers.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the field of molecular biology identification technology, and relates to a biomarker detection reagent, kit, and application for detecting gene mutations in colorectal cancer. Background Technology

[0002] For understanding the technical content of this invention: Colorectal cancer, also known as colon cancer, is a malignant tumor that occurs in the mucosal epithelium of the colon and rectum, and is a common malignant tumor of the digestive tract. Traditional diagnosis and treatment models for colorectal cancer, centered on symptom assessment, imaging examinations, and pathological staging, are insufficient to adequately address the individual differences in the large patient population. Targeted therapy has become an important means of personalized treatment for colorectal cancer. Because the efficacy of targeted therapy is closely related to the tumor's gene status, conducting drug-related gene testing in colorectal cancer patients has significant clinical value in evaluating and predicting the efficacy of targeted therapy.

[0003] Currently, the mainstream technologies for tumor gene status detection include first-generation sequencing (Sanger), next-generation sequencing (NGS), digital PCR (ddPCR), and quantitative PCR (qPCR). Among these, Sanger sequencing has relatively low sensitivity and is prone to missing low-frequency mutations; ddPCR has high single-sample consumable costs and low instrument availability; while NGS can detect even lower-frequency mutations and more mutation types, its experimental procedures are cumbersome and time-consuming, making rapid result delivery difficult. In contrast, qPCR technology is simple to operate, has a short procedure, and extremely high instrument availability. It can simultaneously cover multiple target sites, detect mutation abundance as low as 1%, and its consumable costs are significantly lower than ddPCR. Therefore, qPCR technology is more suitable for widespread implementation in hospitals and can be considered the mainstream technology choice for tumor gene detection.

[0004] Relevant patent documents retrieved: The country of origin for this publication is China, publication number CN120366454A, publication date July 25, 2025. The publication title is "A Primer-Probe Set, Kit, and Application for Detecting KRAS, NRAS, and BRAF Genes in Colorectal Cancer." This publication discloses a primer-probe set, kit, and application for detecting KRAS, NRAS, and BRAF genes in colorectal cancer. It includes a primer-probe set for detecting several mutation sites in the KRAS, NRAS, and BRAF genes, including the following mutation sites: KRAS G12A, KRAS G12C, KRAS G12D, KRASG12R, KRAS G12S, KRAS G12V, KRAS G13D, NRAS G12D, NRAS Q61K, NRAS Q61R, and BRAF V600E. This invention can effectively improve the sensitivity and specificity of gene detection, enabling the simultaneous detection of 11 mutation sites in KRAS, NRAS, and BRAF genes in one tube. It not only detects more genes and mutation sites with a wide coverage, but also saves reagents and consumables, has low detection costs, short detection time (1-2 hours), and is easy to operate.

[0005] Relevant non-patent literature retrieved: The journal title is "China Health Standards Management", the article title is "A Study on the Combined Detection of KRAS / NRAS / PIK3CA / BRAF Gene Mutations in Colorectal Cancer Patients in Hulunbuir Region", volume number 2025, 16(04):119-123, and the publication date is February 15, 2016. This article discloses the preparation of sections of tumor tissue obtained from tumor resection surgery of enrolled patients, and the detection of KRAS / NRAS / PIK3CA / BRAF gene mutations by fluorescence quantitative polymerase chain reaction (PCR) using specific primer double amplification technology.

[0006] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: Currently, most colorectal cancer quantitative PCR detection kits focus on multi-site detection of single genes, with products covering multiple genes and sites being relatively scarce. In clinical practice, however, combined detection of multiple genes is a core basis for targeted therapy drug selection, helping patients find more suitable treatment plans. Existing technologies lack methods to comprehensively detect colorectal cancer gene mutations while ensuring high sensitivity and specificity, which is detrimental to medication and prognosis. While patent literature CN109112226A detects KRAS, NRAS, and BRAF genes in colorectal cancer, its coverage of genes and sites is limited, and the kit's sensitivity and specificity are also limited: at a sample concentration of 1000 copies / mL, the positive detection rate is only 95%; and at a mutation frequency of 5%, mutation results are difficult to detect.

[0007] Therefore, there is an urgent need in this field for a product or method that can detect colorectal cancer gene mutations more comprehensively while ensuring high sensitivity and high specificity. Summary of the Invention

[0008] The purpose of this invention is to provide: A biomarker detection reagent, kit, and application for colorectal cancer gene mutation detection, and related technologies, to solve the technical problem that existing technologies cannot simultaneously achieve comprehensive site coverage, high sensitivity, high specificity, and high accuracy.

[0009] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] Definitions of standard terms can be found in the references “Molecular Cloning: A Laboratory Manual (3rd Edition), Science Press, authors: J. Sambrook and DW Russell, 2002.11”, “Modern Molecular Biology (5th Edition), Higher Education Press, authors: Zhu Yuxian, Li Yi, Zheng Xiaofeng and Guo Hongwei, 2019-06-19”, and “Genetic Engineering, Higher Education Press, 2013-08-01”.

[0012] Unless otherwise stated, conventional methods within the scope of the art, such as flow cytometry, real-time quantitative PCR (qPCR), eukaryotic transcriptome analysis, and cell differentiation identification, shall be used.

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0015] The term "colorectal cancer" used in this article refers to colorectal cancer, which includes colon cancer and rectal cancer.

[0016] The term "target gene" as used in this article refers to the target gene that the reagent or kit is used to specifically detect.

[0017] The term "internal standard gene" used in this article refers to a conserved gene that is co-amplified with the target gene in the detection system. It is used to control the integrity of sample DNA, extraction efficiency, and the effectiveness of the PCR reaction system, ensuring the authenticity and reliability of the detection results.

[0018] The term "fluorescent labeling" as used in this article refers to a technique that uses fluorescent substances (or fluorophores / fluorescent dyes) to specifically label target molecules (such as proteins, nucleic acids, antibodies, etc.) or cell structures, so as to achieve visualization, localization, tracking and quantitative analysis of the target by detecting the fluorescent signals emitted by them.

[0019] The term "limit of detection" as used in this article refers to the lowest concentration or amount of an analyte that an analytical method can reliably detect in a sample under defined experimental conditions.

[0020] As used in this article, "primer" refers to a synthetically produced short oligonucleotide, typically 18-25 bases in length. In a PCR reaction, the primer binds complementary to a specific region of the template DNA, providing a starting point for DNA polymerase to extend the DNA.

[0021] As used in this article, the term "probe" refers to an oligonucleotide fragment labeled with both a fluorescent and a quenching group. In TaqMan quantitative PCR, the probe specifically binds to the target sequence between the primer and the target. During PCR amplification, the 5'→3' exonuclease activity of Taq DNA polymerase cleaves the probe, separating the fluorescent and quenching groups and releasing a fluorescent signal. The intensity of this signal reflects the amplification of the target DNA.

[0022] The term "blocker" as used in this article refers to a specially modified oligonucleotide whose 3' end is blocked (e.g., by adding an MGB group or a C3 spacer), preventing extension in a PCR reaction. Its sequence perfectly matches the wild-type template, and its Tm value is higher than that of the upstream primer. In the reaction, the blocking primer preferentially binds to the wild-type template, preventing non-specific amplification of the upstream primer, thereby significantly improving the specificity of the detection system for mutant templates, especially suitable for samples with a high wild-type background.

[0023] The term "amplification-restricted mutant system (ARMS)" as used in this article refers to allele-specific PCR, also known as allele-specific PCR. This method utilizes the lack of 3'→5' exonuclease activity in Taq DNA polymerase. Specific primers are designed to have their 3' terminal nucleotides complementary to either a mutant or wild-type template. During PCR, primers that perfectly match the template can extend and amplify effectively, while primers with mismatches cannot extend, thus enabling the detection of mutation sites. This invention uses ARMS-PCR technology, taking advantage of the lack of 3'→5' exonuclease activity in Taq DNA polymerase. Allele-specific extension is controlled through 3' terminal primer design, and fluorescence signal values ​​are detected using TaqMan probes to distinguish between wild-type and mutant alleles. By designing the 3' terminal nucleotides of the upstream primers for alleles to be identical to the mutant site and different from the wild-type site, under the action of Taq DNA polymerase, upstream primers that do not perfectly match the negative template will not form complete complementary base pairs, resulting in mismatches and no PCR product. Conversely, primer systems that match the positive template can amplify the corresponding PCR product. A TaqMan probe is placed between the forward and reverse primers. During PCR amplification, Taq DNA polymerase, with its 5'→3' exonuclease activity, cleaves the probe, and the fluorescent group on the probe generates a fluorescent signal that can be detected by the instrument. ARMS primers (amplification-restricted mutant system primers) can distinguish between mutant and wild-type templates. They are designed to amplify only mutant templates, while having very weak or no amplification ability for wild-type templates.

[0024] The term “XNA modification” used in this article refers to: Xenonucleic acid, which is an artificial analog developed by modifying the three basic components of natural nucleic acids (furanose, phosphodiester backbone and bases) to achieve chemical modification by replacing the deoxyribose structure in DNA.

[0025] In a first aspect, the present invention provides: a biomarker detection reagent for detecting gene mutations in colorectal cancer, the biomarker detection reagent comprising a combination of amplification primers for detecting mutation sites in KRAS, NRAS, PIK3CA and BRAF genes, the combination of amplification primers comprising primers for amplifying blocked mutation systems and ordinary primers; The nucleotide sequences of the primers for the amplification blocked mutation system include those shown in SEQ ID NO:1-35; The nucleotide sequences of the common primers include those shown in SEQ ID NO:41-49.

[0026] According to some embodiments of the present invention, the biomarker detection reagent further includes a probe assembly, the nucleotide sequence of which includes the sequence shown in SEQ ID NO:50-58.

[0027] According to some embodiments of the present invention, the biomarker detection reagent further includes a blocking primer assembly, the nucleotide sequence of which includes the sequence shown in SEQ ID NO:36-40.

[0028] According to some embodiments of the present invention, at least one nucleotide in the blocking primer combination is an XNA-modified nucleotide.

[0029] XNA modification is selected from at least one of LNA (locked nucleus), FANA (2'-fluoroarabinonucleotide), HNA (hexitol nucleic acid), TNA (threononucleotide), ANA (arabinonucleotide), CeNA (cyclohexene nucleic acid), and PNA (peptide nucleic acid). LNA is preferred for XNA modification.

[0030] According to some embodiments of the present invention, the 3' end of each sequence in the blocking primer combination is blocked, such as by adding an MGB group or a C3 spacer.

[0031] According to some embodiments of the present invention, the biomarker detection reagent further includes an internal standard primer and an internal standard probe for detecting the internal reference gene HER2, wherein the nucleotide sequence of the internal standard primer includes the sequence shown in SEQ ID NO:60-61 or a sequence having at least 85% sequence identity with the shown sequence; and the nucleotide sequence of the internal standard probe includes the sequence shown in SEQ ID NO:59 or a sequence having at least 85% sequence identity with the shown sequence.

[0032] Furthermore, the nucleotide sequence of the internal standard primer includes the sequence shown in SEQ ID NO:60-61 or a sequence having at least 90% sequence identity with the shown sequence.

[0033] Furthermore, the nucleotide sequence of the internal standard primer includes the sequence shown in SEQ ID NO:60-61 or a sequence having at least 95% sequence identity with the shown sequence.

[0034] Furthermore, the nucleotide sequence of the internal standard primer includes the sequence shown in SEQ ID NO:60-61 or a sequence having at least 99% sequence identity with the shown sequence.

[0035] Preferably, the nucleotide sequence of the internal standard primer includes the sequence shown in SEQ ID NO:60-61.

[0036] For example, the nucleotide sequence of the internal standard primer has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or any value within the range of any two of the above values ​​as shown in SEQ ID NO:60-61.

[0037] Furthermore, the nucleotide sequence of the internal standard probe includes the sequence shown in SEQ ID NO:59 or a sequence having at least 90% sequence identity with the shown sequence.

[0038] Furthermore, the nucleotide sequence of the internal standard probe includes the sequence shown in SEQ ID NO:59 or a sequence having at least 95% sequence identity with the shown sequence.

[0039] Furthermore, the nucleotide sequence of the internal standard probe includes the sequence shown in SEQ ID NO:59 or a sequence having at least 99% sequence identity with the shown sequence.

[0040] Preferably, the nucleotide sequence of the internal standard probe includes the sequence shown in SEQ ID NO:59.

[0041] For example, the nucleotide sequence of the internal standard probe includes the sequence shown in SEQ ID NO:59 having sequence consistency of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any value within the range of any two of the above values.

[0042] According to some embodiments of the present invention, the nucleotide sequences of the probe assembly and the internal standard probe are respectively equipped with a fluorescent reporter group and a quencher group at both ends. The fluorescent reporter group is selected from ROX, FAM or VIC (HEX), and the quencher group is selected from BHQ1 and BHQ2.

[0043] Preferably, the probes used to detect mutation sites in the KRAS and BRAF genes are labeled with FAM fluorescent markers, the probes used to detect mutation sites in the NRAS and PIK3CA genes are labeled with ROX fluorescent markers, and the internal standard probes used to detect internal standard genes are labeled with VIC (HEX) fluorescent markers.

[0044] According to some embodiments of the present invention, the mutation sites include: 19 somatic mutations in exons 2-4 of the KRAS gene, 12 somatic mutations in exons 2-3 of the NRAS gene, 5 somatic mutations in exons 9 and 20 of the PIK3CA gene, and V600E mutation in exon 15 of the BRAF gene, that is, 37 mutation sites.

[0045] Specifically, the 19 somatic mutations in exons 2-4 of the KRAS gene include: KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS A146T, KRAS A146P, KRAS K117N(A>T), KRAS Q61R, KRAS A146V, KRAS A59T, KRAS G12S, KRAS G12A, KRAS G13D, KRAS G13C, KRAS Q61H(A>T), KRAS Q61L, KRAS K117N(A>C), KRAS Q61H(A>C), and KRAS Q61K.

[0046] Specifically, the 12 somatic mutations in exons 2-3 of the NRAS gene include: NRAS G12R, NRAS Q61H (A>C), NRAS Q61H (A>T), NRAS Q61R, NRAS Q61K, NRAS Q61L, NRAS G12D, NRAS G12V, NRAS G13R, NRAS G12C, NRAS G13V, and NRAS G13D.

[0047] Specifically, the five somatic mutations in exons 9 and 20 of the PIK3CA gene include: PIK3CA E542K, PIK3CAH1047R, PIK3CA H1047L, PIK3CA E545K, and PIK3CA E545D (G>T).

[0048] According to some embodiments of the present invention, the marker detection reagent can be stored or used in the form of lyophilized powder or solution dissolved in buffer.

[0049] Secondly, the present invention provides a kit for detecting gene mutations in colorectal cancer, the kit comprising the aforementioned biomarker detection reagents.

[0050] Furthermore, the kit also includes a reaction apparatus, which can be a PCR tube. PCR tubes include single tubes and multi-tube arrays, preferably 8-tube arrays.

[0051] Preferably, in the kit, the biomarker detection reagents are pre-dispensed into the reaction wells of the reaction device in a specific combination, and the biomarker detection reagents and PCR tubes form a reaction strip for easy use.

[0052] According to some embodiments of the present invention, the kit further includes at least one of PCR reaction solution, positive control and negative control.

[0053] Preferably, the PCR reaction solution may include Taq DNA polymerase, dNTPs, and Mg. 2+ and at least one of the reaction buffer.

[0054] Preferably, the positive control is selected from positive plasmid DNA or cell lines containing the above-mentioned mutation sites; the negative control is selected from wild-type DNA without mutation sites, sterile water or purified water.

[0055] Furthermore, the kit also includes a kit instruction manual.

[0056] According to some embodiments of the present invention, the method for detecting biomarkers for colorectal cancer gene mutation detection using the kit includes the following steps: S1, Obtain the DNA sample of the biological sample to be tested; S2, The DNA sample is tested using the kit, and fluorescence signals are collected; S3, determine whether the biological sample to be tested has a corresponding mutation based on the fluorescence signal.

[0057] According to some embodiments of the present invention, the detection of the DNA sample using the kit in step S2 includes performing a qPCR reaction on the DNA sample using the kit. The qPCR is preferably multiplex qPCR. Further, the qPCR reaction is at least partially an amplification arrestor mutant system PCR (ARMS-PCR). Even further, all qPCR reactions are performed using ARMS-PCR.

[0058] For example, the qPCR reaction system may be: PCR reaction solution, 10 μL-15 μL; single primer, 30 pmol-100 pmol; single probe, 10 pmol-20 pmol; single blocker, 0 pmol-40 pmol; template DNA, 2 μL-8 μL; purified water, to a final volume of 25 μL. It is understood that the specific components and their amounts in the above-described qPCR reaction system are only one preferred embodiment. Those skilled in the art, based on the kit disclosed in this invention, can routinely optimize or adjust the total volume of the reaction system (e.g., from 25 μL to 50 μL), the types, concentrations, and volumes of components in the reaction system according to actual detection needs, the instruments and equipment used, and the performance of the reagents. These obvious modifications are all within the scope of this invention.

[0059] For example, the qPCR reaction procedure is shown in Table 1.

[0060] Table 1

[0061] According to some embodiments of the present invention, the specific interpretation criteria for determining whether the biological sample to be tested has a corresponding mutation based on the fluorescence signal in step S3 are shown in Table 2.

[0062] Table 2

[0063] Fourthly, the present invention provides the application of the above-mentioned biomarker detection reagent or the above-mentioned kit in the preparation of products for the detection of colorectal cancer gene mutations, wherein the application is for non-disease diagnosis and treatment purposes.

[0064] The non-disease diagnosis and treatment purposes include at least one of the following: scientific research, drug screening, or industrial applications (manufacturing, assembly, or quality control of reagent kits).

[0065] Specifically, scientific research may include: research on the gene mutation mechanism of colorectal cancer, mutation spectrum analysis of mutated genes, etc.; drug screening may include: screening or evaluation of anti-tumor drugs; reagent kit quality control may include: quality testing for batch-to-batch consistency of reagent kits during the production process.

[0066] In this invention, Example 1 at least supports the protection scope of "marker detection reagent for colorectal cancer gene mutation detection".

[0067] The term "biomarker detection reagent for colorectal cancer gene mutation detection" is derived from the foregoing explanation and / or the corresponding reagents in Example 1. Therefore, those skilled in the art can reasonably presume that "biomarker detection reagent for colorectal cancer gene mutation detection," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of "biomarker detection reagent for colorectal cancer gene mutation detection."

[0068] Example 1 of this invention at least supports the protection scope of "a kit for detecting gene mutations in colorectal cancer".

[0069] The term "kit for detecting gene mutations in colorectal cancer" is derived from the foregoing explanation and / or the corresponding kit in Example 1. Therefore, those skilled in the art can reasonably presume that "kit for detecting gene mutations in colorectal cancer," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of "kit for detecting gene mutations in colorectal cancer."

[0070] The present invention has at least the following beneficial effects: Compared with existing technologies, this invention provides a biomarker detection reagent, kit, and application for colorectal cancer gene mutation detection, which has better technical effects, specifically in the following aspects: (1) The biomarker detection reagent of the present invention, based on the realization of four-gene joint detection and broad-spectrum coverage of 37 mutations, has specifically optimized and validated the primer system for KRAS G12C mutations of currently approved targeted drugs (such as KRAS G12C inhibitors). These targeted drugs effectively block the transmission of downstream oncogenic signaling pathways and inhibit tumor cell proliferation by specifically binding to KRAS G12C protein and locking its inactive state, thus significantly improving the clinical efficacy (tumor shrinkage or disease stabilization) of some patients. Based on this, the biomarker detection reagent can accurately detect KRAS G12C mutation sites and provide clear detection results while completing four-gene joint detection, providing technical support for the clinical application of these targeted drugs and having higher clinical applicability and prospectiveness.

[0071] (2) The biomarker detection reagent of the present invention, through the unique design of primers for the amplification-blocked mutation system and ordinary primers, enables the present invention to generate no false positive signals in the detection of 100 ng wild-type background DNA, providing a signal-to-noise ratio guarantee that surpasses the traditional ARMS technology for the stable detection of low-frequency mutations (1%), and has superior specificity and sensitivity. Furthermore, for the blocking of wild-type template, the present invention also uses an XNA (heterologous nucleic acid) modified blocker. Compared with conventional phosphorylation blocking blockers, XNA modified blockers have higher Tm values ​​and stronger nuclease resistance, can form a more stable blocking complex with wild-type template, and cannot be recognized by DNA polymerase, further improving the specificity of the biomarker detection reagent of the present invention.

[0072] (3) In this invention, the conserved region of the human HER2 gene is selected as the internal standard to design internal standard primers and internal standard probes. After adjusting and testing different amplification fragments of the HER2 gene, the final determined amplification region can not only effectively monitor the extraction quality of sample DNA and PCR inhibitors, but also more realistically reflect the fragmentation degree of FFPE sample DNA, ensuring the reliability of the detection results.

[0073] (4) This invention integrates a detection system targeting up to 37 mutations in four genes—KRAS, NRAS, PIK3CA, and BRAF—into a single 8-tube PCR reaction tube, achieving a simplified operation mode of "one-step sample addition, full gene coverage." This greatly reduces the risk of sample confusion caused by multi-tube operations and is more suitable for the high-throughput, standardized testing scenarios in hospital laboratories. At the same time, by adopting a two-step amplification strategy, the entire detection time is shortened to less than 75 minutes, significantly improving detection efficiency. Attached Figure Description

[0074] Figure 1 Example 3 of the present invention uses the biomarker detection reagent and kit from Example 1 to detect the BRAF gene positive amplification map of clinical sample No. 5.

[0075] Figure 2 Example 3 of the present invention uses the biomarker detection reagent and kit from Example 1 to detect the positive amplification of BRAF gene and PIK3CA gene in clinical sample No. 6.

[0076] Figure 3 In Example 3 of the present invention, the marker detection reagent and kit in Example 1 were used to detect the negative amplification map of clinical sample No. 10.

[0077] Figure 4 Example 3 of the present invention uses the biomarker detection reagent and kit from Example 1 to detect the KRAS gene positive amplification map of clinical sample No. 21.

[0078] Figure 5 Example 3 of the present invention uses the biomarker detection reagent and kit from Example 1 to detect the NRAS gene positive amplification map of clinical sample No. 43. Detailed Implementation

[0079] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0080] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0081] Data analysis and statistical analysis were performed using professional data processing software, and significance analysis was conducted using one-way ANOVA. P <0.05 indicates a significant difference.

[0082] The main instrument used in the following examples is a real-time PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd., SLAN-96S).

[0083] Example 1: Biomarker detection reagents and kits for colorectal cancer gene mutation detection The biomarker detection reagent includes the ability to detect 37 mutation sites in the KRAS, NRAS, PIK3CA, and BRAF genes (KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS A146T, KRAS A146P, KRAS K117N(A>T), KRAS Q61R, KRAS A146V, KRAS A59T, KRAS G12S, KRAS G12A, KRAS G13D, KRAS G13C, KRAS Q61H(A>T), KRAS Q61L, KRAS K117N(A>C), KRAS Q61H(A>C), KRAS Q61K, NRAS G12R, NRAS Q61H(A>C), NRAS Q61H(A>T), NRAS Q61R, NRAS Q61K, NRAS...). Amplification primer combinations for Q61L, NRAS G12D, NRAS G12V, NRASG13R, NRAS G12C, NRAS G13V, NRAS G13D, PIK3CA E542K, PIK3CA H1047R, PIK3CA H1047L, PIK3CA E545K, PIK3CA E545D(G>T), and BRAF V600E were used. These primer combinations included primers for amplifying blocked mutation systems and ordinary primers. The nucleotide sequences of the primers for amplifying blocked mutation systems were as shown in SEQ ID NO:1-35, and the nucleotide sequences of the ordinary primers were as shown in SEQ ID NO:41-49.

[0084] The biomarker detection reagent also includes a probe assembly, the nucleotide sequence of which includes the sequence shown in SEQ ID NO:50-58. The biomarker detection reagent also includes a blocking primer assembly, the nucleotide sequence of which includes the sequence shown in SEQ ID NO:36-40. The biomarker detection reagent also includes an internal standard primer and an internal standard probe for detecting the internal reference gene HER2, the nucleotide sequence of which includes the sequence shown in SEQ ID NO:60-61; and the nucleotide sequence of which includes the sequence shown in SEQ ID NO:59.

[0085] The components of the kit are shown in Table 3.

[0086] Table 3

[0087] In Example 1, the biomarker detection reagent was pre-allocated to 8-tube PCR units. Specific information about the above sequences and their positions in the wells of the 8-tube PCR units are shown in Table 4.

[0088] Table 4

[0089] Table 4 (continued 1)

[0090] Table 4 (Continued 2)

[0091] Note: "F" represents the upstream primer, and "R" represents the downstream primer.

[0092] As shown in Table 4, after extensive primer sequence design and site combination testing, Example 1 of this invention simultaneously detected 37 mutation types in a limited number of 8 wells, with the most mutation types detected in one well, along with one internal control gene site, further improving the sensitivity and specificity of detection. Furthermore, since KRAS and NRAS genes have relatively high sequence similarity, primer design often suffers from poor specificity. This invention employs precise sequence design to effectively distinguish between KRAS and NRAS detection, and can further improve the distinguishing effect through special primer modifications.

[0093] Comparative Example 1 The only difference between the biomarker detection reagent in Comparative Example 1 and Example 1 is that the ARMS primers, regular primers, and probes for detecting the KRAS gene are all replaced; the components of the remaining biomarker detection reagents are the same as in Example 1. The components of the kit in Comparative Example 1 are essentially the same as in Example 1, except for the biomarker detection reagents.

[0094] The biomarker detection reagents were pre-allocated to 8-tube PCR tubes. Except for the ARMS primers, regular primers, and probes corresponding to the KRAS gene, the pre-allocation positions of the remaining ARMS primers, regular primers, probes, internal control primers, and internal control probes remained unchanged. Please refer to Table 5 for specific information on the above sequences and their placement in the 8-tube PCR tubes.

[0095] Table 5

[0096] Comparative Example 2 The only difference between the biomarker detection reagent in Comparative Example 2 and Example 1 is that some of the ARMS primers for detecting the 8 wells were replaced; the remaining components of the biomarker detection reagent are the same as in Example 1. The components of the kit in Comparative Example 2 are essentially the same as in Example 1, except for the biomarker detection reagent. For detailed information on the sequence of the marker detection reagent for Comparative Example 2 and the well positions in the 8-tube PCR aliquots in the kit, please refer to Table 6.

[0097] Table 6

[0098] Example 1: Detection Limit Assessment Preparation of 1% Reference Samples and Digital PCR Calibration: Genomic DNA from the negative cell line GM12878 (purchased from Shanghai Haling Biotechnology Co., Ltd., catalog number HLXC1039) was used as a matrix and mixed with 37 positive plasmids for mutation sites at a copy number ratio of 1:99 to construct reference samples with an expected mutation frequency of 1%. Using a Sinaf digital PCR instrument, the above reference sample samples were calibrated and detected according to the reaction system (Table 7) and procedure (Table 8) described below. The detection results of Example 1 are shown in Table 9. Analysis revealed that the actual mutation frequency of all reference samples was approximately 1%, necessitating further quantitative real-time PCR detection.

[0099] Table 7

[0100] Table 8

[0101] Table 9

[0102] (1) Real-time PCR amplification: Prepare the reaction mixture according to the reaction system in Table 10. Take 5 μL of the test reference sample (diluted with purified water and measured to a concentration of 2 ng / μL using a Qubit fluorometer), positive control, and negative control, and add them to the corresponding reaction wells in the 8-tube array. Carefully cap the reaction tubes, perform a brief centrifugation, and then conduct quantitative real-time PCR detection according to the reaction procedure set in Table 11. During the detection process, collect fluorescence signals from the FAM, ROX, and VIC / HEX channels simultaneously.

[0103] Table 10

[0104] Table 11

[0105] (2) Analysis of test results: After the reaction was completed, the baseline and threshold were manually or automatically adjusted according to the PCR instrument manual and the fluorescence curve to obtain the Ct value of each sample.

[0106] Quality control: Negative controls ①-⑧ have no Ct values ​​in the FAM and ROX channels, and the internal standard VIC / HEX channel has no S-shaped amplification curve. Occasionally, a slight rise in the amplification curve may occur, which is normal and does not affect the interpretation of the test results. Positive controls have S-shaped amplification curves in the FAM, ROX, and VIC / HEX channels, and each well is interpreted as positive according to Table 2. All of the above conditions must be met in the same experiment; otherwise, the results of this experiment will be invalid.

[0107] Positive cutoff value: CT < 35 for internal standard in sample. The determination of each gene is based on the ΔCt value of FAM, ROX channels and internal standard VIC / HEX channels in wells ①-⑧ of reaction strips, as detailed in Table 2.

[0108] Referring to Table 12, in this experiment, the detection results of both the negative and positive control samples from Example 1 met the preset quality control standards; according to the interpretation criteria, the ΔCt value of the 1% concentration reference sample was less than the threshold, indicating a positive result. Therefore, this demonstrates that the limit of detection (LOD) for the 37 detection sites is 1%, and the reagent of this invention can still detect some sites with mutation abundance below 1% as verified by digital PCR.

[0109] Table 12

[0110] Using the same experimental procedure, the marker primers, probes, and reagents of Comparative Example 1 and Comparative Example 2 were used to detect the aforementioned 37 positive mutation reference samples. The detection results of Comparative Example 1 are shown in Table 13: After replacing the ARMS primers, regular primers, and probes for the KRAS gene, the Ct values ​​of the KRAS gene and other sites in the same well in all 8 wells increased significantly; and digital PCR verification showed that for sites with mutation abundance below 1%, the ΔCt exceeded the interpretation threshold, resulting in false negative results. The detection results of Comparative Example 2 are shown in Table 14: After replacing some sites of the ARMS primers in the 8 wells, the replaced sites and other sites in the same well were affected, and the Ct values ​​all increased. The changes in the target sites of the replaced ARMS primers were more pronounced, and false negatives occurred at some sites. Based on the above results, it is found that the primer sequences finally determined by this invention after extensive primer combination screening and optimization are the optimal combinations.

[0111] Table 13

[0112] Table 14

[0113] Example 2: Specificity Test 100 ng of negative cell line GM12878 DNA was selected as the test sample, and a specificity detection experiment was carried out according to the reaction system and amplification procedure of Example 1. The specificity judgment criteria were: a negative result was defined as ΔCt (target Ct value - internal reference Ct value) greater than the interpretation threshold; the larger the ΔCt value, the better the specificity; if there was no obvious amplification curve of the target, it was the best specificity performance.

[0114] Results Analysis: Please refer to Table 15. By comparing and analyzing the test data of marker primers, probes and reagents in Example 1 and Comparative Examples 1-2, using 100 ng negative cell lines as samples, the results show that Example 1 has the best specificity, and no amplification signal (NoCt) was found in any 100 ng negative sample.

[0115] Table 15

[0116] Example 3: Testing of 50 clinical samples Using the detection reagents, reaction system, and procedure described in this invention, a validation experiment was conducted on 50 paraffin-embedded tissue samples from colorectal cancer (samples were obtained from Weinan Central Hospital, with informed consent signed by the patients, and all samples were tested using next-generation sequencing (NGS) technology to obtain corresponding results). Specific experimental details are as follows: Extraction of DNA from 50 colorectal cancer tissue samples: DNA extraction was performed using the Migi Bio Magnetic Bead FFPE DNA Extraction Kit (D6323), detailed in the kit instructions. An RNase digestion process was added during extraction to prevent residual RNA from affecting concentration determination. The extracted DNA concentration was determined using a Qμbit fluorometer; the DNA concentration should be ≥2 ng / μL, and the total extraction volume should not be less than 80 ng. Before testing, the DNA in the samples was diluted with purified water to a concentration of 2 ng / μL, in a volume greater than 40 μL.

[0117] The steps, quality control analysis, and interpretation methods for quantitative real-time PCR amplification were described in Example 1. Table 16 shows the interpretation results of 50 clinical samples detected using the biomarker detection reagents and kits described in Example 1. Amplification images of some of the detection results are shown below. Figures 1 to 5 As shown.

[0118] Table 16 Clinical Sample Test Results

[0119] Note: "+" represents positive, and "-" represents negative.

[0120] Using next-generation sequencing (NGS) results from 50 clinical samples as a reference, a consistency analysis was performed on the detection performance of the kit of this invention. Please refer to Table 17. Statistical analysis shows that, compared to comparative examples 1-2, the positive and negative concordance rates of the kit results in Example 1 and the reference results both reached 100%, with an overall consistency of 100%. The above data fully demonstrates that the kit of this invention significantly improves detection efficiency, shortens the detection cycle, and enhances sensitivity and specificity, while covering multiple gene loci and ensuring the accuracy of detection results, thus possessing performance advantages for clinical application.

[0121] Table 17

[0122] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A biomarker detection reagent for detecting gene mutations in colorectal cancer, characterized in that, The biomarker detection reagent includes a combination of amplification primers for detecting mutation sites in the KRAS, NRAS, PIK3CA, and BRAF genes, wherein the amplification primer combination includes primers for amplifying blocked mutation systems and ordinary primers; The nucleotide sequences of the primers for the amplification blocked mutation system include those shown in SEQ ID NO:1-35; The nucleotide sequences of the common primers include those shown in SEQ ID NO:41-49.

2. The biomarker detection reagent according to claim 1, characterized in that, The biomarker detection reagent also includes a probe assembly, the nucleotide sequence of which includes the sequence shown in SEQ ID NO:50-58.

3. The biomarker detection reagent according to claim 1, characterized in that, The biomarker detection reagent also includes a blocking primer set, the nucleotide sequence of which includes the sequence shown in SEQ ID NO:36-40.

4. The biomarker detection reagent according to claim 3, characterized in that, At least one nucleotide in the blocking primer combination is an XNA-modified nucleotide.

5. The biomarker detection reagent according to claim 1, characterized in that, The biomarker detection reagent further includes an internal standard primer and an internal standard probe for detecting the internal reference gene HER2. The nucleotide sequence of the internal standard primer includes the sequence shown in SEQ ID NO:60-61 or a sequence having at least 85% sequence identity with the shown sequence. The nucleotide sequence of the internal standard probe includes the sequence shown in SEQ ID NO:59 or a sequence having at least 85% sequence identity with the shown sequence.

6. The biomarker detection reagent according to claim 1, characterized in that, The mutation sites include: 19 somatic mutations in exons 2-4 of the KRAS gene, 12 somatic mutations in exons 2-3 of the NRAS gene, 5 somatic mutations in exons 9 and 20 of the PIK3CA gene, and the V600E mutation in exon 15 of the BRAF gene.

7. A kit for detecting gene mutations in colorectal cancer, characterized in that, The kit includes the biomarker detection reagent as described in any one of claims 1-6.

8. The reagent kit according to claim 7, characterized in that, The kit also includes at least one of PCR reaction solution, positive control and negative control.

9. The reagent kit according to claim 7, characterized in that, The method for detecting biomarkers for colorectal cancer gene mutation detection using the kit includes the following steps: S1, Obtain the DNA sample of the biological sample to be tested; S2, The DNA sample is tested using the kit, and fluorescence signals are collected; S3, determine whether the biological sample to be tested has a corresponding mutation site based on the fluorescence signal.

10. The use of the biomarker detection reagent of any one of claims 1-6 or the kit of any one of claims 7-9 in the preparation of a product for detecting gene mutations in colorectal cancer, wherein the use is for non-disease diagnosis and treatment purposes.

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