A fluorescence in situ hybridization combined detection probe for HER2 gene and FGFR2 gene and application thereof

Abstract

The present application relates to a kind of HER2 gene and FGFR2 gene fluorescence in situ hybridization combined detection probe and its application, belong to biological medicine technical field.The present application provides a kind of probe and probe composition for tumor diagnosis and prognosis evaluation, including the first probe for detecting HER2 gene, and the second probe and the third probe for detecting FGFR2 gene;First probe, second probe and third probe are respectively marked with the first fluorochrome, second fluorochrome and third fluorochrome that can produce different colors;By the present application, a kind of fluorescence in situ hybridization combined detection method and its kit for simultaneously detecting HER2 and FGFR2 gene variation are provided;Wide application range can be used in various tissue and cell samples needing simultaneously detecting HER2 and FGFR2 gene state, one detection, two results, meet the goal of clinical test quality improvement and efficiency improvement;It has quite extensive application prospect.

Description

Technical Field

[0001] This invention relates to a fluorescence in situ hybridization detection probe for the HER2 gene and the FGFR2 gene and its application, belonging to the field of biomedical technology. Background Technology

[0002] The FGFR2 gene, located at 10q26.13, plays a crucial role in human development, growth, and maintenance. Its full name is Fibroblast Growth Factor Receptor 2. The protein encoded by the FGFR2 gene is a receptor tyrosine kinase. Located on the cell surface, it receives signals from extracellular fibroblast growth factors (FGFs). FGFs, acting as ligands, specifically bind to the FGFR2 protein on the cell surface, leading to a conformational change in the FGFR2 receptor, typically forming a dimer (two receptor molecules binding). Dimerization activates the tyrosine kinase activity in the intracellular region of the receptor. The activated kinase phosphorylates specific tyrosine residues (adding a phosphate group). The phosphorylated tyrosine residues become the activation signal for downstream signaling molecules, activating a series of downstream signaling pathways (such as the RAS / MAPK, PI3K / AKT, PLCγ, and STAT pathways). These pathways ultimately transmit signals to the cell nucleus, regulating the expression of specific genes and thus affecting cellular function. The FGFR2 signaling pathway is crucial for many life processes, such as embryonic development, especially in skeletal development (formation and patterning of the skull, facial bones, and limb bones) and organogenesis (formation of organs such as the lungs, skin, inner ear, and brain). It guides cell proliferation, differentiation, migration, and survival. During tissue repair and regeneration, it participates in wound healing, angiogenesis (new blood vessel formation), and bone repair. It maintains the normal structure and function of certain tissues (such as skin, bone, and prostate) to ensure tissue homeostasis. It also participates in phosphate metabolism and vitamin D regulation (related to bone health).

[0003] Abnormalities in the FGFR2 gene (primarily gain-of-function mutations, i.e., mutations leading to overactivation or persistent activation of the receptor) are closely associated with a variety of diseases. For example, skeletal developmental syndromes, including Apert syndrome, are the most typical and severe, caused by mutations at two specific sites (S252W, P253R) on the FGFR2 gene. Affected children exhibit pyramidal head (pointed head), midfacial hypoplasia (facial depression), syndactyly (fusion of fingers or toes), and intellectual development may be affected. Crouzon syndrome is also relatively common, caused by different mutations in the FGFR2 gene. It manifests as craniosynostosis leading to various cranial deformities (brachycephaly, triangular head, etc.), exophthalmos, and midfacial hypoplasia. It is usually not accompanied by severe syndactyly. Pfeiffer syndrome is caused by mutations in FGFR1 and FGFR2 (FGFR2 mutations are more common). Characteristic features include craniosynostosis, wide and misaligned thumb and big toe, partial syndactyly, midfacial hypoplasia, and exophthalmos. Jackson-Weiss syndrome is rare, primarily affecting the skull and foot. Beare-Stevenson gyrate skin syndrome is extremely rare, characterized by gyrate skin (deep grooves), craniosynostosis, and acanthosis nigricans. Some cases of Antley-Bixler syndrome are caused by FGFR2 mutations, characterized by craniosynostosis, radius-humerus bony junction, joint contractures, and genitourinary abnormalities. More seriously, FGFR2 gene abnormalities (including point mutations, gene amplification, and gene fusion / rearrangement) are driving factors for various cancers, especially in the following tumors: 1. Gastric cancer: FGFR2 amplification or overexpression is one of the important molecular subtypes of gastric cancer (especially diffuse gastric cancer), associated with poor prognosis, and is an important target for targeted therapy. The incidence of FGFR2 gene translocation is low (approximately 1%-3%), with translocation types such as FGFR2-TACC3 and FGFR2-CIT. 2. Breast cancer: FGFR2 amplification is present in some breast cancers (especially hormone receptor-positive breast cancers). FGFR2 gene translocations have been detected in some triple-negative breast cancers (TNBC). Translocated genes include FGFR2-TACC2 and FGFR2-KIAA1598. 3. FGFR2 mutations are relatively common in endometrial cancer. FGFR2 gene translocations are reported in very few cases. 4. FGFR2 translocations or amplifications can be seen in non-small cell lung cancer, such as fusions like FGFR2-KIAA1967. 5. Cholangiocarcinoma: FGFR2 translocation is an important targetable driver gene alteration in cholangiocarcinoma (especially intrahepatic cholangiocarcinoma). FGFR2 translocation is the most common therapy-sensitive variant in intrahepatic cholangiocarcinoma, accounting for approximately 15%-20%. Common fusion partner genes include BICC1, AHCYL1, CCDC6, and PPL. 6. Salivary gland carcinoma: FGFR2 translocations are occasionally seen in pleomorphic adenomas or carcinomas.Due to the driving role of FGFR2 in various cancers, the development of targeted drugs against the FGFR signaling pathway (especially FGFR2) has been a hot topic in recent years, such as small molecule tyrosine kinase inhibitors and pan-FGFR inhibitors (which simultaneously inhibit FGFR1, 2, 3, and 4). Examples include erdatinib (approved for urothelial carcinoma with FGFR2 / 3 fusion / rearrangement), pemitinib (approved for cholangiocarcinoma with FGFR2 fusion / rearrangement), inflavinib (cholangiocarcinoma), fobatinib (cholangiocarcinoma), ropetinib (cholangiocarcinoma), and derazantinib.

[0004] Therefore, the function and regulatory mechanisms of the FGFR2 gene and its encoded protein are of great significance for understanding human developmental biology, diagnosing genetic diseases, and developing precision therapies for related cancers.

[0005] Methods for detecting FGFR2 include FISH, NGS (RNA sequencing is preferred), or PCR to detect fusion variants. FISH, an in situ tissue assay, is a commonly used gene detection method that can detect FGFR2 gene translocations and amplifications. In tumors such as gastric cancer, breast cancer, and cholangiocarcinoma, HER2 gene amplification also occurs frequently, and its amplification status is often detected in these tumors. Therefore, this invention combines the FGFR2 and HER2 genes to develop a probe that simultaneously detects both genes, improving detection efficiency. Summary of the Invention

[0006] One of the technical problems to be solved by this invention is how to prepare a dual-gene simultaneous fluorescence in situ hybridization joint detection probe for the HER2 gene and the FGFR2 gene and its application.

[0007] One of the technical problems to be solved by this invention is how to prepare a detection kit for HER2 and FGFR2 gene abnormalities and the preparation method thereof. The kit contains probes that target specific chromosomal loci or combinations of multiple specific chromosomal loci, which can rapidly and effectively detect a variety of cancer samples. It is suitable for the development and promotion of cancer diagnosis and prognostic assessment products; it provides reference information for clinical molecular targeted therapy and is conducive to the development of individualized treatment strategies for patients in clinical practice.

[0008] To address the aforementioned problems, the present invention provides a molecular marker for tumor diagnosis and prognostic assessment. This molecular marker is a combination of the HER2 and FGFR2 genes. The HER2 gene is located at Chr17: 39,688,094-39,728,658; the FGFR2 gene is located at Chr10: 121,478,330-121,598,458. The data are sourced from NCBI.

[0009] The present invention provides a probe and probe composition for tumor diagnosis and prognostic assessment, the probe and probe composition being used to detect molecular markers as described above, including a first probe for detecting the HER2 gene, and a second and third probe for detecting the FGFR2 gene; the first, second and third probes are respectively labeled with a first fluorescein, a second fluorescein and a third fluorescein that can produce different colors.

[0010] Preferably, the fluorescence colors of the first fluorescein, the second fluorescein, and the third fluorescein are red, green, and cyan, respectively; the first fluorescein, the second fluorescein, and the third fluorescein are respectively selected from tetramethylrhodamine, fluorescein isothiocyanate, and cyanin.

[0011] The present invention provides the use of the above-described molecular markers, or the above-described probes and probe compositions, in the preparation of a detection kit for tumor diagnosis and prognostic assessment.

[0012] This invention provides a detection kit for tumor diagnosis and prognostic assessment. The kit is used to detect human chromosomal abnormalities. The detected chromosomal loci include the HER2 gene and the FGFR2 gene. The detection kit contains a set of fluorescent in situ hybridization probes for detecting the chromosomal loci. The target fragment of the fluorescent in situ hybridization probe set for detecting the HER2 gene is Chr17: 39,688,094-39,728,658; the target fragment of the fluorescent in situ hybridization probe set for detecting the FGFR2 gene is Chr10: 121,478,330-121,598,458.

[0013] Preferably, the test kit further includes a sample collection device and consumables; the sample collection device collects samples from blood, saliva, urine, pleural effusion, or ascites.

[0014] Preferably, the test kit further includes several test reagents, which include at least one of digestion solution, washing solution, sample preservation solution, or organic reagent for FISH hybridization.

[0015] The present invention provides the use of the above-described detection kit in the preparation of tumor diagnostic or prognostic products.

[0016] The present invention provides the use of the above-described detection kit in the preparation of gene detection products, the use including for detecting rearrangement / translocation of the FGFR2 gene, amplification of the FGFR2 gene and / or amplification of the HER2 gene.

[0017] This invention provides a detection system comprising a data processing device and a substance for detecting biomarkers. The data processing device includes a data input module, a data recording module, a data comparison module, and a conclusion output module. The substance for detecting biomarkers includes probes, probe compositions, and detection reagents as described above. The data input module is configured to input a biomarker detection image of a sample to be tested. The data recording module is configured to store the sample detection image and a judgment threshold. The data comparison module is configured to receive the biomarker detection image of the sample to be tested sent by the data input module, and retrieve the judgment threshold from the data recording module and compare it with the sample detection image. The conclusion output module is configured to receive the comparison result sent by the data comparison module, and judge the comparison result according to predetermined judgment conditions to determine the genetic abnormality of the tested individual.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. This invention provides a fluorescence in situ hybridization (FISH) method and kit for simultaneously detecting FGFR2 and HER2 gene variants. The kit has a wide range of applications and can be used for various tissue and cell samples requiring simultaneous detection of FGFR2 and HER2 gene status, such as tumor cells, exfoliated cells from pleural and peritoneal fluid, and normal cells.

[0020] 2. The detection method provided by this invention is simple and efficient, meeting the needs of rapid clinical diagnosis. A single test yields multiple results, aligning with the goal of improving the quality and efficiency of clinical laboratory testing. This invention provides abundant clinical pathological and prognostic information, demonstrating high clinical application value for disease prognosis prediction.

[0021] 3. This invention uses three colors for intensive labeling, achieving the goal of detecting two genes simultaneously on a single slice in one operation.

[0022] 4. This invention saves on small biopsy tissue samples and allows for the screening of two molecular targets in a single test. Attached Figure Description

[0023] Figure 1 A diagram showing FGFR2 gene abnormalities in pan-tumor regions, based on TCGA data;

[0024] Figure 2This is a graph showing the proportion of FGFR2 gene abnormalities in various tumors in TCGA data.

[0025] Figure 3 This is a graph showing the proportion of HER2 gene abnormalities in various tumors in TCGA data.

[0026] Figure 4 This is a schematic diagram of the probe design of the present invention;

[0027] Figure 5 This is a fluorescence in situ hybridization (FISH) image of cells with abnormal FGFR2 gene (separation of green and cyan signals of FGFR2 gene, with an increased copy number of cyan signal) and unamplified HER2 gene (red signal) in biliary system tumor cells.

[0028] Figure 6 This is a fluorescence in situ hybridization (FISH) image of biliary tract tumor cells showing abnormal FGFR2 gene (separation of green and cyan signals) and unamplified HER2 gene (red signal).

[0029] Figure 7 This is a fluorescence in situ hybridization (FISH) image of HER2 gene amplification (red signal) in biliary system tumor cells. Detailed Implementation

[0030] To make the present invention more apparent and understandable, preferred embodiments are described in detail below:

[0031] Example 1

[0032] Preparation of a FISH probe for detecting cellular gene status, tumor malignancy, and prognosis:

[0033] This embodiment provides a FISH probe combination for detecting cellular gene status, tumor malignancy, and prognosis, involving probes for detecting the FGFR2 and HER2 genes, such as... Figure 4 The detection probes for the FGFR2 and HER2 genes are labeled with fluorophores that produce different fluorescent colors.

[0034] 1. The preparation steps for HER2 gene and FGFR2 gene probes are as follows:

[0035] (1) BAC clone screening: Clones containing HER2 and FGFR2 gene sequences were screened and purchased from the Invitrogen RP11 BAC clone library. BAC clones targeting the HER2 gene are shown in Table 1 below, and BAC clones targeting the FGFR2 gene are shown in Tables 2 and 3 below. These BAC clones were referenced to the genome version Human Feb. 2009 (GRCh37 / hg19).

[0036] Table 1

[0037]

[0038] Table 2

[0039]

[0040] Table 3

[0041]

[0042] Plasmid extraction: Using a commercially available plasmid extraction kit, plasmids were extracted from the BAC clones according to the kit instructions to obtain plasmid DNA, which was then quantified using Nanodrop 2000.

[0043] Plasmid DNA fluorescent labeling: The plasmid DNA was fluorescently labeled using a nick-shifting method. For the HER2 gene detection probe, a mixture of two fragments as shown in Table 1 was used for fluorescent labeling, with tetramethylrhodamine (TRITC) being the preferred fluorescein. For the FGFR2 gene fragment 1 detection probe, a mixture of five fragments as shown in Table 2 was used for fluorescent labeling, with Cyanine being the preferred fluorescein. For the FGFR2 gene fragment 2 detection probe, a mixture of three fragments as shown in Table 3 was used for fluorescent labeling, with fluorescein isothiocyanate (FITC) being the preferred fluorescein. The PCR reaction system was prepared on ice under strictly dark conditions. The probe labeling reaction system is shown in Table 4 below.

[0044] Table 4

[0045]

[0046] After the system is prepared, shake to mix and centrifuge, label at 25°C for 2 hours, and incubate at 80°C for 10 minutes to inactivate the enzyme.

[0047] The labeled product was precipitated with sodium acetate, centrifuged at high speed, and then purified. The probe was dissolved in purified water to obtain the labeled GSP (Gene Special Probe) HER2 gene probe and GSP FGFR2 gene probe one / GSP FGFR2 gene probe two.

[0048] Example 2

[0049] Kit for detecting the gene status of biliary system tumor cells:

[0050] 2.1 This embodiment provides a detection kit for the gene status of biliary system tumor cells, enabling direct observation of signals in tissue samples. The kit contains a hybridization solution of the FISH probe combination described in Example 1. The hybridization solution includes probes, a hybridization buffer solution, placental DNA, and in situ hybridization blue staining solution.

[0051] The hybridization buffer contains sodium citrate buffer (SSC), deionized formamide, and dextran sulfate (DSS), with the concentration of deionized formamide being 40%-60% and the concentration of DSS being 0.1-0.2 g / mL. The in situ hybridization blue staining solution is 4',6-diamidinyl-2-phenylindole (DAPI).

[0052] The composition and preparation of the hybridization solution for the kit are shown in Table 5 below.

[0053] Table 5

[0054]

[0055] 2.2 FISH Testing Procedure

[0056] 2.2.1 Section Pretreatment:

[0057] 1) Use a rotary microtome to cut 3-4μm tissue samples onto a 40-50℃ water surface. After spreading the slides, retrieve them onto a glass slide to prevent them from falling off. Place the glass slides in an 80±5℃ constant temperature oven for 45 minutes.

[0058] 2) After baking, the slices are immersed in xylene I and II in sequence at room temperature for 10 minutes to dewax; then they are immersed in 100% and 90% graded ethanol for 3 minutes each; and washed with water for 3 minutes.

[0059] 3) Remove the slices and wash them in sterile purified water at room temperature for 3 minutes;

[0060] 4) Remove the slides and boil them in antigen retrieval buffer at 100°C for 20 minutes. Remove the slides and air dry at room temperature.

[0061] 5) Place the slices in preheated pepsin digestion solution (proteinase K working solution (200μg / ml) at 37±1℃ and digest for 5-10 minutes; stop digestion with distilled water; dry at 37℃.

[0062] 2.2.2 Add the probe (operation in the dark)

[0063] 1) Take the hybridization probes of HER2 and FGFR2 gene 1 / FGFR2 gene 2 out of the -20℃ freezer, shake to mix, and centrifuge briefly; add 10μl of hybridization solution to the hybridization area, quickly cover with a 10×10mm coverslip, remove air bubbles, and seal the edges with rubber glue.

[0064] 2) Place the glass slide on the heating plate of the in situ hybridization instrument, place the absorbent strip pre-soaked in double-distilled water into the slot on the hybridization instrument cover, close the hybridization instrument cover, and set the program: 85℃ for 5-10 minutes, hybridize overnight at 37℃.

[0065] 2.2.3 Washing and counterstaining after hybridization (operation in the dark)

[0066] 1) Remove the slide, gently peel off the rubber, remove the coverslip, and incubate in 2×SSC at 37±1℃ for 10 minutes;

[0067] 2) Remove the slice and incubate it in 0.1% NP-40 / 2×SSC at 37±1℃ for 5 minutes;

[0068] 3) Remove the slide and place it in 70% ethanol at room temperature for 3 minutes to dehydrate; remove the slide and allow it to air dry in the dark.

[0069] 4) Add 10 μL of DAPI counterstain to the hybridization area of ​​the slide at room temperature, cover with a coverslip, and observe under a microscope after 15 minutes.

[0070] 2.2.4 Observation of FISH results using fluorescence microscopy:

[0071] 1) Scan the entire slide under a 40× objective lens. A satisfactory specimen should have hybridization signals in more than 75% of the cancer cell nuclei; observe whether heterogeneity exists.

[0072] 2) Locate a clear tumor area under a 100× objective lens, observe the FISH staining results of the tumor cell nuclei, and count the signal.

[0073] 2.3 Detection of HER2 and FGFR2 gene status in biliary system tumor cells:

[0074] Methods and Results: Between 2024 and 2025, the Department of Pathology at Zhongshan Hospital Affiliated to Fudan University detected FGFR2 gene rearrangements in 167 cases, of which 157 were biliary tract tumors and 10 were metastatic biliary tract tumors. 109 cases were hepatobiliary surgical resection specimens, and 58 cases were small biopsy specimens (34.7%).

[0075] Between 2024 and 2025, 621 cases were found to have HER2 gene amplification, including 198 cases of gastric cancer, 178 cases of breast cancer, 143 cases of colorectal cancer, 68 cases of pancreatic and biliary tract origin (including 56 cases of gallbladder, intrahepatic bile duct and extrahepatic bile duct, 8 cases of pancreatic duct adenocarcinoma, and 4 cases of duodenal ampullary carcinoma), as well as urothelial carcinoma, ovarian cancer, salivary gland tumors, etc.

[0076] Description of the attached drawings: Figure 1 TCGA data showed that FGFR2 gene abnormalities exist in pan-tumorous diseases.

[0077] Figure 2 This is a graph showing the proportion of FGFR2 gene abnormalities in various tumors in TCGA data.

[0078] Figure 3 This is a graph showing the proportion of HER2 gene abnormalities in various tumors from TCGA data.

[0079] Figure 4 This is a schematic diagram of the probe design of the present invention; wherein, the HER2 gene is marked in red, and the copy number of the HER2 gene can be counted based on the red signal. The FGFR2 gene is marked in green and cyan, and whether the green and cyan signals are separated can be used to determine whether the FGFR2 gene has undergone translocation; the copy number of the FGFR2 gene can be counted based on the cyan signal.

[0080] Figure 5 This is a fluorescence in situ hybridization (FISH) image of biliary tract tumor cells. In these cells, the FGFR2 gene shows separate green and cyan signals, with an increased copy number of the cyan signal (>4), suggesting FGFR2 gene rearrangement accompanied by an increased FGFR2 gene copy number. The HER2 gene (red signal) copy number is not increased.

[0081] Figure 6 This is a fluorescence in situ hybridization (FISH) image of biliary tract tumor cells. In the biliary tract tumor cells, the green and cyan signals of the FGFR2 gene are separated, suggesting FGFR2 gene rearrangement. The copy number of the HER2 gene (red signal) is not increased.

[0082] Figure 7 This is a fluorescence in situ hybridization (FISH) image of biliary tract tumor cells. In these cells, the increased red signal of the HER2 gene suggests HER2 gene amplification.

[0083] This invention provides a fluorescence in situ hybridization method for detecting FGFR2 gene translocation and HER2 gene amplification, which can assist in the molecular subtyping of cancer and potential targeted therapies, thereby helping to achieve more accurate and effective diagnosis of cancer molecular subtypes, screening potential targeted therapy populations, providing reference information for clinical molecular targeted therapy, and facilitating the development of individualized treatment strategies for patients.

[0084] This invention can simultaneously detect FGFR2 gene translocation and HER2 gene amplification, such as in tumor cells, exfoliated cells from pleural and peritoneal fluid, and normal cells.

[0085] The detection method of this invention is simple and efficient, and can meet the needs of rapid clinical diagnosis. One test yields two results, which meets the goal of improving quality and efficiency.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A molecular biomarker for tumor diagnosis and prognostic assessment, characterized in that, The molecular markers are a combination of the HER2 and FGFR2 genes; the HER2 gene is located at Chr17: 39,688,094-39,728,658; the FGFR2 gene is located at Chr10: 121,478,330-121,598,458; the above data are from NCBI.

2. A probe and probe composition for tumor diagnosis and prognostic assessment, characterized in that, The probe and probe composition are used to detect the molecular markers as described in claim 1, including a first probe for detecting the HER2 gene, and a second and third probe for detecting the FGFR2 gene; the first, second, and third probes are respectively labeled with a first fluorophore, a second fluorophore, and a third fluorophore that can produce different colors.

3. The probe and probe composition for tumor diagnosis and prognostic assessment according to claim 2, characterized in that, The fluorescence colors of the first fluorescein, the second fluorescein, and the third fluorescein are red, green, and cyan, respectively; the first fluorescein, the second fluorescein, and the third fluorescein are respectively tetramethylrhodamine, fluorescein isothiocyanate, and cyanin.

4. The use of the molecular marker according to claim 1, or the probe and probe composition according to any one of claims 2-3, in the preparation of a detection kit for tumor diagnosis and prognostic assessment.

5. A diagnostic kit for tumor diagnosis and prognostic assessment, characterized in that, The kit is used to detect human chromosomal abnormalities; the chromosomal loci to be detected include the HER2 gene and the FGFR2 gene, and the kit contains a set of fluorescent in situ hybridization probes for detecting the chromosomal loci; wherein, the target fragment of the fluorescent in situ hybridization probe set for detecting the HER2 gene is Chr17: 39,688,094-39,728,658; and the target fragment of the fluorescent in situ hybridization probe set for detecting the FGFR2 gene is Chr10: 121,478,330-121,598,458.

6. The detection kit according to claim 5, characterized in that, The test kit also includes a sample collection device and consumables; the sample collection device collects samples from blood, saliva, urine, pleural effusion, or peritoneal effusion; the test kit also includes several test reagents, including at least one of digestive fluid, washing solution, sample preservation solution, or organic reagents for FISH hybridization.

7. Use of the test kit according to any one of claims 5-6 in the preparation of tumor diagnostic or prognostic products.

8. The use of the detection kit according to any one of claims 5-6 in the preparation of gene detection products, characterized in that, The applications include detecting rearrangements / translocations of the FGFR2 gene, amplification of the FGFR2 gene, and / or amplification of the HER2 gene.

9. A detection system, characterized in that, The system includes a data processing device and a substance for detecting biomarkers. The data processing device includes a data input module, a data recording module, a data comparison module, and a conclusion output module. The substance for detecting biomarkers includes probes, probe compositions, and detection reagents as described in claims 2-3. The data input module is configured to input a biomarker detection image of a sample to be tested. The data recording module is configured to store the sample detection image and a judgment threshold. The data comparison module is configured to receive the biomarker detection image of the sample to be tested sent by the data input module, and retrieve the judgment threshold from the data recording module and compare it with the sample detection image. The conclusion output module is configured to receive the comparison results sent by the data comparison module, and to determine the comparison results according to predetermined judgment conditions to determine the genetic abnormality of the tested person.

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