Method, system, kit and probe for detecting BRCA2 gene mutation

Abstract

The invention relates to the technical field of molecular detection, in particular to a method, a system, a kit and a probe for detecting BRCA2 gene mutation. The detection method comprises the following steps: carrying out pretreatment and PCR amplification on a blood sample, carrying out a hybridization reaction with a probe to obtain a sample, detecting the sample based on an SERS technology, and collecting an SERS spectrum. Whether BRCA2 gene mutation exists or not is judged by identifying characteristic double peaks of a cy3 fluorophore at 1188 cm <-1 > and 1393 cm <-1 >, and the whole detection process can be completed within 2 hours. According to the scheme, the region, carrying the BRCA2 gene, on the DNA can be amplified through the specific primer pair, then the DNA is specifically combined through the improved oligonucleotide probe, and detection is achieved through the reporter molecule on the oligonucleotide probe; therefore, the detection method provided by the scheme has the advantages of high detection speed, high sensitivity and high detection result accuracy, and the problems of long time consumption and high cost of the traditional PCR or NGS technology are effectively solved.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology, specifically to a method, system, kit, and probe for detecting BRCA2 gene mutations. Background Technology

[0002] BRCA2 (Breast Cancer Susceptibility Protein 2) is an important tumor suppressor gene located in the 13q13.1 region of human chromosomes. The protein it encodes participates in the homologous recombination pathway for DNA doublet break repair, which is crucial for maintaining genomic stability. Furthermore, the protein encoded by the BRCA2 gene works synergistically with molecules such as BRCA1 and RAD51 to ensure proper repair of DNA damage, thereby preventing malignant transformation of cells. Pathogenic mutations in the BRCA2 gene (such as frameshift mutations and nonsense mutations) lead to loss of protein function, resulting in the accumulation of DNA damage, increased genomic instability, and consequently, a significantly increased risk of developing various malignant tumors, including breast cancer and ovarian cancer.

[0003] In hereditary breast cancer, BRCA2 mutation carriers have a significantly increased lifetime risk of developing breast cancer, ranging from 40% to 70%, and are also associated with earlier age of onset and an increased risk of bilateral breast cancer. In ovarian cancer, approximately 10% to 15% of cases are associated with BRCA2 gene mutations, and BRCA2 mutation carriers have a lifetime risk of developing the disease of approximately 10% to 30%. Furthermore, BRCA2 gene mutations are also associated with an increased risk of male breast cancer, pancreatic cancer, and prostate cancer. Therefore, determining the presence of BRCA2 gene mutations is of significant value in risk assessment, prevention, intervention, and treatment strategies. In terms of risk assessment, BRCA2 gene testing for high-risk individuals with a family or personal history of breast / ovarian cancer can clarify their genetic susceptibility and achieve individualized risk stratification. Regarding prevention, BRCA2 mutation carriers can adopt more proactive surveillance strategies (such as earlier and more frequent breast MRI scans) or consider preventative surgeries (such as bilateral mastectomy or salpingo-oophorectomy) to significantly reduce the risk of related cancers. In treatment decisions, BRCA2 mutation status can influence the choice of surgical approach (such as contralateral prophylactic resection). More importantly, for patients with advanced breast or ovarian cancer, poly-ADP ribose polymerase inhibitors (PARPi) can specifically target the DNA repair defects in BRCA-mutated cells, exerting a "synthetic lethality" effect, and have become an important targeted therapy.

[0004] Currently, the main methods used clinically to detect BRCA2 gene mutations include Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and next-generation sequencing (NGS). Among them, Sanger sequencing has high accuracy but low throughput and long processing time, making it difficult to meet the needs of large-fragment gene screening; MLPA can effectively detect large fragment deletions / duplications, but cannot identify point mutations or small fragment variations; NGS can detect whole-genome sequences with high throughput, but data analysis is complex, costly, and has certain requirements on sample quality and tumor cell content. Summary of the Invention

[0005] To address the technical problems of poor accuracy and low sensitivity in existing methods for detecting BRCA2 gene mutations, this invention provides a method, system, kit, and probe for detecting BRCA2 gene mutations.

[0006] This invention employs the following technical solution: a method for detecting BRCA2 gene mutations, comprising: performing PCR amplification on a blood sample to be tested using a primer pair with the upstream primer having the nucleotide sequence of SEQ ID NO.2 and the downstream primer having the nucleotide sequence of SEQ ID NO.3; co-incubating the amplified PCR product with an oligonucleotide probe having the nucleotide sequence of SEQ ID NO.1 and a cy3 fluorescent group attached to its 5' end to obtain a sample; and detecting the sample at 1188 cm⁻¹ using SERS technology. -1 and 1393cm -1 Does the sample contain a characteristic peak at 1188 cm⁻¹? Make the following judgment: -1 and 1393cm -1 If characteristic peaks are present at all locations, it indicates that the blood sample to be tested contains a BRCA2 gene mutation; otherwise, it indicates that the blood sample to be tested does not contain a BRCA2 gene mutation.

[0007] As a further improvement of the present invention, the blood sample to be tested needs to be pretreated as follows before PCR amplification: obtain the blood sample, and add cell lysis buffer and biomembrane lysis buffer to the peripheral blood sample in sequence to lyse it. After lysis, add DNA adsorption solution, and then wash with detergent to obtain the blood sample to be tested.

[0008] As a further improvement of the present invention, the cell lysis buffer is a mixture of protein lyase K and PBS at a volume ratio of 1:9.

[0009] As a further improvement of the present invention, the biofilm lysis buffer is Lysis Buffer.

[0010] As a further improvement of the present invention, the DNA adsorption solution is anhydrous ethanol.

[0011] As a further improvement of the present invention, the detergent includes ddH2O and a washing buffer; the washing buffer is a mixture of 80% formamide and 5% EDTA in a volume ratio of 2:1.

[0012] As a further improvement of the present invention, the washing agent is used for washing in the following manner: the blood sample after adding DNA adsorption solution is first washed multiple times with ddH2O. Then, a washing buffer with a volume ratio of 5-6:25 to ddH2O is added to the washed blood sample for further washing, thereby obtaining the pretreated blood sample.

[0013] As a further improvement of the present invention, in the pretreatment of blood samples, the volume ratio of cell lysis buffer to blood sample and the volume ratio of biolysis buffer to blood sample are both 1:2. The volume ratio of DNA adsorption buffer to blood sample is 1:10 to 1:20.

[0014] As a further improvement to this invention, the SERS technology employs a Raman spectrometer for detection. Before detection, the sample must be dropped onto a substrate and allowed to dry. The excitation wavelength of the Raman spectrometer is 780nm-790nm, the excitation power is 25mW-30mW, and the integration time is 500ms.

[0015] As a further improvement of the present invention, the concentration of the oligonucleotide probe is 8 μM.

[0016] As a further improvement of the present invention, the volume ratio of the amplified PCR product to the oligonucleotide probe during co-incubation is 50:1.

[0017] As a further improvement of this invention, the substrate is a silver nanoparticle substrate. The preparation method of the silver nanoparticle substrate is as follows: A suspension of spherical silver nanoparticles and citric acid is centrifuged at 6500 r / min to precipitate the spherical nanoparticles, and the supernatant is removed. Then, ddH2O is added to the precipitated spherical silver nanoparticles to bring the volume to 80 times the volume of the spherical silver nanoparticles before centrifugation, resulting in a second mixture. 5 μL of this second mixture is added dropwise to a silicon wafer and dried at room temperature to obtain the silver nanoparticle substrate.

[0018] This invention also provides a system for detecting BRCA2 gene mutations, which employs the aforementioned method for detecting BRCA2 gene mutations. The system includes a blood processing module, a Raman spectrometer, and a data processing module. The blood processing module sequentially pre-processes, amplifies, co-incubates, and dries the blood sample to be tested, thereby obtaining the sample to be tested. The Raman spectrometer acquires the molecular vibrational / rotational information of the sample to be tested and generates a Raman spectrum. The data processing module has a built-in 1188cm² sensor. -1 and 1393cm -1A dual-peak identification algorithm. The data processing module acquires Raman spectra and makes the following judgment: if the sample's Raman spectrum is above 1188 cm⁻¹... -1 and 1393cm -1 If a characteristic peak is present at all locations, it indicates that the blood sample to be tested has a BRCA2 gene mutation and outputs "mutation positive"; otherwise, it indicates that the blood sample to be tested does not have a BRCA2 gene mutation and outputs "mutation negative".

[0019] The present invention also provides a kit for storing a pre-treated blood sample to be tested in the method for detecting BRCA2 gene mutations as described above.

[0020] This invention also provides a probe, which is an oligonucleotide probe used in the method for detecting BRCA2 gene mutations as described above. The probe is used to bind to the BRCA2 gene sequence when a BRCA2 gene mutation occurs, thereby capturing the BRCA2 gene.

[0021] The technical solution provided by this invention has the following beneficial effects: (1) The method for detecting BRCA2 gene mutations provided by this invention improves the oligonucleotide probe by linking the cy3 fluorescent group to the 5' end of the oligonucleotide, thereby enabling the binding of the cy3 reporter molecule to the oligonucleotide. When the BRCA2 gene is mutated, the improved oligonucleotide probe can bind its nucleotide sequence to the BRCA2 sequence, thereby capturing the BRCA2 gene. At the same time, the oligonucleotide probe carrying the cy3 group can serve as a reporter molecule, and its characteristic peak is at 1188 cm⁻¹. -1 and 1393cm -1 Therefore, based on this principle, during detection, it can be observed that the distance is 1188cm. -1 and 1393cm -1 The presence of a characteristic peak simultaneously determines whether a BRCA2 gene mutation has occurred. This detection principle is simple, and the dual-peak detection method further eliminates false positives that can occur during single-peak detection, thus improving the accuracy of the entire detection method. Furthermore, this method uses SERS technology to detect BRCA2 gene mutations, effectively improving detection sensitivity and speed. The detection time provided by this method can be reduced to 2 hours, the consistency between the detection results and gene sequencing results is greater than 100%, and the detection sensitivity can reach a mutation frequency greater than 0.1%, effectively solving the problems of long processing times and high costs associated with traditional PCR or NGS technologies.

[0022] (2) The method for detecting BRCA2 gene mutations provided in this scheme can amplify trace amounts of nucleic acids (such as the BRCA2 gene) in DNA through amplification operations, thereby raising the level of extremely small amounts or even single amounts of nucleic acids to detectable and analyzable levels. With the help of special primer pairs, the specific primer pairs can amplify the regions on DNA that carry the target gene (i.e., the BRCA2 gene), thereby rapidly screening out fragments on DNA that carry the target gene (BRCA2 gene) and amplifying these fragments, thereby improving the specificity and sensitivity of the detection and effectively shortening the detection time. Then, the improved oligonucleotide probes specifically bind to the DNA, and the detection is achieved through the reporter molecules on the oligonucleotide probes. Thus, the detection method provided in this scheme has the advantages of fast detection speed, high sensitivity, and high accuracy of detection results. Attached Figure Description

[0023] Figure 1 This is a flowchart of the steps of the method for detecting BRCA2 gene mutations provided in Embodiment 1 of the present invention.

[0024] Figure 2 These are the SERS spectra of Examples 5 to 7 of the present invention.

[0025] Sequence List Description (Sequence list content provided separately) SEQ ID NO.1 is the nucleotide sequence of the oligonucleotide probe in this embodiment of the invention; SEQ ID NO.2 is the DNA nucleotide sequence of the upstream primer in this embodiment of the invention; SEQ ID NO.3 is the DNA nucleotide sequence of the downstream primer in this embodiment of the invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] Example 1 This embodiment provides a method for detecting BRCA2 gene mutations. Please refer to [link / reference]. Figure 1 It includes: (a) The blood sample to be tested is amplified by PCR using primer pairs.

[0028] In this scheme, the primer pair consists of the upstream primer with the nucleotide sequence of SEQ ID NO.2 and the downstream primer with the nucleotide sequence of SEQ ID NO.3.

[0029] 1.1 Obtain the blood sample to be tested The blood sample to be tested needs to be pretreated before PCR amplification using primer pairs. The specific pretreatment process is as follows: Obtain the blood sample, and add cell lysis buffer and biomembrane lysis buffer sequentially to lyse the peripheral blood sample. After lysis, add DNA adsorption buffer, and then wash with detergent to obtain the blood sample to be tested.

[0030] The blood sample obtained can be a peripheral blood sample. The cell lysis buffer can be a mixture of protein lyase K and PBS (phosphate buffer) at a volume ratio of 1:9. The purpose of adding the cell lysis buffer is to lyse the cells in the blood sample. The biomembrane lysis buffer can be Lysis Buffer. The added biomembrane lysis buffer is used to lyse and break the cell membrane and nuclear membrane, thereby releasing the genomic DNA in the cells, which facilitates better adsorption of the DNA in the cells by the subsequently added DNA adsorbent. The DNA adsorbent can be anhydrous ethanol. The washing agent can include ddH2O and washing buffer; the washing buffer is a mixture of 80% formamide and 5% EDTA at a volume ratio of 2:1.

[0031] During the pretreatment of blood samples, the volume ratio of cell lysis buffer to blood sample and the volume ratio of biolysis buffer to blood sample were both 1:2; the volume ratio of DNA adsorption buffer to blood sample was 1:10 to 1:20.

[0032] The washing process is as follows: the blood sample after adding DNA adsorption solution is first washed multiple times with ddH2O; then, a washing buffer with a volume ratio of 5-6:25 to ddH2O is added to the washed blood sample for further washing, thus obtaining the pre-treated blood sample.

[0033] The following is a specific pretreatment procedure for the blood sample to be tested: Take 100-200 μL of peripheral blood sample from a breast cancer patient. Add 50-100 μL of cell lysis buffer to the peripheral blood sample, mix by pipetting, and let stand for 3 min. After adding cell lysis buffer, mix by pipetting, and let stand for 3 min, add another 50-100 μL of biomembrane lysis buffer, and mix the peripheral blood sample with the added biomembrane lysis buffer by pipetting again. Place a silica membrane in a 2 mL EP tube as an adsorption column, and then place the biolyzed peripheral blood sample in the adsorption column and add 10-20 μL of DNA adsorption buffer. The added DNA adsorption buffer allows the released DNA to be specifically adsorbed onto the adsorption column. Wash twice with ddH2O, and then centrifuge at 16000g for 5 min after washing to remove impurities such as proteins and RNA from the cells and obtain relatively pure DNA. Finally, 100-120 μL of washing buffer was added to the centrifuged blood sample to wash it, thereby eluting relatively pure DNA from the adsorption column. After washing, the sample was centrifuged at 1000g for 5 minutes to remove as much washing buffer as possible, thus improving DNA elution efficiency and yield.

[0034] 1.2 PCR amplification Add 1 μL of pretreated blood sample to the PCR system. The primer pair in the PCR system consists of a 100 μM upstream primer and a 100 μM downstream primer. The nucleotide sequence of the upstream primer is 5'-GCT GTT TCT TCA GGATGG CTG A-3'. The nucleotide sequence of the downstream primer is 5'-CAC TGC ACA CCA CCA TCA TCA-3'. The amplification parameters can be set as follows: initialization temperature can be 95℃, time can be 5 min; the number of cycles can be 45; during the cycle, the denaturation phase temperature can be 95℃, time can be 30 s; during the cycle, the annealing phase temperature can be 55℃, time can be 50 s; during the cycle, the extension phase temperature can be 72℃, time can be 45 s; and after the cycle, the final extension phase temperature can be 72℃, time can be 10 min. For specific amplification procedures, refer to existing techniques. The above amplification operation can amplify trace amounts of nucleic acids (such as the BRCA2 gene) in DNA, enabling the amplification of extremely small amounts, or even single quantities, of nucleic acids to a detectable and analyzable level. With the help of specific primer pairs, which can amplify regions on DNA that carry the target gene (i.e., the BRCA2 gene), fragments carrying the target gene (BRCA2 gene) can be quickly screened from DNA and amplified. This improves the specificity and sensitivity of the detection and effectively shortens the detection time.

[0035] (ii) Binding the amplified PCR product with an oligonucleotide probe The oligonucleotide probe has the nucleotide sequence SEQ ID NO.1, specifically: aac cca gct gtg tggccg. Its 5' end is linked to a cy3 fluorescent group. The oligonucleotide concentration can be 8 μM, and it should be stored at -20°C protected from light. The volume ratio of the oligonucleotide probe to the amplified PCR product during co-incubation can be 1:50.

[0036] It is understandable that oligonucleotides can be used as probes on their own. In this scheme, the oligonucleotide probe is improved by attaching the cy3 fluorescent group to the 5' end of the oligonucleotide, thus enabling the binding of the cy3 reporter molecule to the oligonucleotide. When the BRCA2 gene is mutated, the improved oligonucleotide probe's nucleotide sequence can bind to the BRCA2 sequence, thereby capturing the BRCA2 gene. Simultaneously, the cy3 group carried by the oligonucleotide probe can serve as a reporter molecule, and its characteristic peak is at 1188 cm⁻¹. -1 and 1393cm -1 Therefore, based on this principle, during detection, it can be observed that the distance is 1188cm. -1 and 1393cm -1 The presence or absence of characteristic peaks is used to determine whether a mutation has occurred in the BRCA2 gene. The cy3 group can be ligated to the 5' end of the oligonucleotide probe using an automated DNA synthesizer.

[0037] The following is a specific procedure for binding amplified PCR products with oligonucleotide probes: Take 20 μL of PCR product and add it to 0.4 μL of oligonucleotide probe, incubate at 95℃ for 5 min to anneal and bind, and then obtain the sample.

[0038] (III) Sample detection based on SERS technology This method is based on SERS technology to detect samples. Therefore, before detecting the samples, the samples need to be dropped onto the substrate and dried at room temperature. Then, the glass slide with the sample dropped onto the substrate is placed in a Raman spectrometer for SERS detection.

[0039] The substrate can be silver nanoparticles, and its preparation process is as follows: A spherical silver nanoparticle suspension with a particle concentration of 6 × 10⁸ particles / mL is centrifuged at 6500 r / min for 10 min to precipitate the nanoparticles, and the supernatant is removed. Then, ddH₂O is added to the precipitated spherical silver nanoparticles to bring the volume to 80 times the original volume. Finally, 5 μL of the diluted solution is added dropwise onto a glass slide and dried at room temperature to obtain a glass slide with a silver nanoparticle substrate. The glass slide can be made of silicon material.

[0040] A small Raman spectrometer, such as the NR785E10W-Plus, can be used. The excitation wavelength can be 785 nm, the detection power 30 mW, and the integration time 500 ms. By using the Raman spectrometer with these parameters, the SERS spectrum of the sample (i.e., the SERS spectrum of the blood sample to be tested) can be obtained. SERS technology possesses high sensitivity, fingerprint recognition capability, and non-destructive testing characteristics. Applying it to the detection of BRCA2 gene mutations enables rapid and low-cost screening for BRCA2 gene mutations, which is of great significance for popularizing genetic screening and guiding clinical baseline prevention and treatment, thus giving the entire detection method broad clinical application prospects.

[0041] By determining the obtained SERS spectrum at 1188 cm⁻¹ -1 and 1393cm -1 The presence of characteristic peaks at 1188 cm⁻¹ is used to determine whether a BRCA2 gene mutation exists in the blood sample being tested. The specific determination process is as follows: If the sample shows a characteristic peak at 1188 cm⁻¹... -1 and 1393cm -1 The presence of characteristic peaks at all 1188 cm⁻¹ indicates the presence of a BRCA2 gene mutation in the blood sample being tested; otherwise, it indicates the absence of a BRCA2 gene mutation in the blood sample. As described above, this method involves observing the peaks at 1188 cm⁻¹ in the SRES spectrum. -1 and 1393cm -1 The presence or absence of characteristic peaks at both points significantly reduces the difficulty of determining whether a BRCA2 gene mutation has occurred compared to relying solely on changes in characteristic peaks. Furthermore, this method improves both the speed and accuracy of the determination. Moreover, by establishing this dual-peak detection standard, this approach effectively avoids the occurrence of single peaks due to detection noise during single-peak determination, thus preventing false positives and ensuring greater accuracy.

[0042] As described in the above implementation steps, the method for detecting BRCA2 gene mutations provided by this scheme first improves the purity of DNA in blood samples through pretreatment, avoiding positive results due to other components in the blood sample and reducing detection errors at the source. Then, a probe linked to a fluorescent group specifically binds to the BRCA2 gene mutation, and loosely bound probes are removed by washing with a washing buffer. Finally, the peak generated by the fluorescent group in the final product is detected by SERS, enhancing the SERS substrate's ability to detect BRCA2 gene mutations and improving the sensitivity and accuracy of SERS technology for detecting BRCA2 gene mutations. Therefore, the detection method provided by this scheme has the characteristics of high sensitivity, rapid detection, and low cost. Specifically, the nucleotide sequence of the probe provided by this scheme is SEQ ID NO.1, which can bind to the BRCA2 gene sequence when a BRCA2 gene mutation occurs, thereby capturing the BRCA2 gene. Combined with the 5'-linked cy3 group of the oligonucleotide probe, this fluorescent reporter molecule allows the sample to be detected at 1188 cm⁻¹ after binding to DNA. -1 and 1393cm -1 The presence or absence of characteristic peaks at both ends determines whether a BRCA2 gene mutation has occurred. This detection principle is simple, and the dual-peak detection method further eliminates false positives that can occur during single-peak detection, thus improving the accuracy of the entire detection method. Furthermore, this method uses SERS technology to detect BRCA2 gene mutations, effectively improving detection sensitivity and speed. The detection time provided by this method can be reduced to 2 hours, the consistency between the detection results and gene sequencing results is greater than 100%, and the detection sensitivity can reach a mutation frequency of up to 0.1%. This effectively solves the problems of long processing times and high costs associated with traditional PCR or NGS technologies, making this method suitable for rapid intraoperative diagnosis and guidance for personalized targeted therapy.

[0043] Understandably, the results of the BRCA2 gene mutation detection method provided in this protocol can offer some guidance for breast cancer prognostic assessment. For example, if the test result is "mutation positive," targeted therapy can be prioritized; if the result is "mutation negative," conventional chemotherapy, such as paclitaxel, can be prioritized. However, in actual breast cancer prognostic assessment, the patient's specific condition and other external factors must be considered, and the test results in this protocol can only serve as one reference indicator for breast cancer prognostic assessment.

[0044] Example 2 This embodiment provides a system for detecting BRCA2 gene mutations based on Embodiment 1, employing the method for detecting BRCA2 gene mutations as described in Embodiment 1. The system includes a blood processing module, a Raman spectrometer, and a data processing module. The blood processing module sequentially pre-processes, amplifies, co-incubates, and dries the blood sample to be tested, thereby obtaining the sample to be tested. The Raman spectrometer acquires the molecular vibrational / rotational information of the sample to be tested and generates a Raman spectrum. The data processing module has a built-in 1188cm... -1 and 1393cm -1 A dual-peak identification algorithm. The data processing module acquires the Raman spectrum from the Raman spectrometer and makes the following judgment: If the Raman spectrum of the sample is at 1188 cm⁻¹... -1 and 1393cm -1 If a characteristic peak is present at all locations, it indicates that the blood sample to be tested contains a BRCA2 gene mutation and outputs "mutation positive"; otherwise, it indicates that the blood sample to be tested does not contain a BRCA2 gene mutation and outputs "mutation negative".

[0045] Example 3 This solution provides a kit based on Example 1, which is used to store the blood sample to be tested after pretreatment in the method for detecting BRCA2 gene mutations as described in Example 1.

[0046] Example 4 This embodiment provides a probe based on Example 1. The probe is an oligonucleotide probe used in the method for detecting BRCA2 gene mutations in Example 1. The probe is used to bind to the BRCA2 gene sequence when a BRCA2 gene mutation occurs, thereby capturing the BRCA2 gene.

[0047] The following specific examples demonstrate that the technical solution provided in this application can effectively detect BRCA2 gene mutations.

[0048] Example 5 Patient Chen XX, female, 62 years old, was diagnosed with breast cancer carrying a BRCA2 gene mutation in June 2025. Peripheral blood was collected from the patient, and a probe was used to detect the BRCA2 gene mutation, including the following steps: 1. Collect blood samples from the patient. 100 μL of peripheral blood was collected from the patient, and cell lysis buffer was added to lyse the cells. Then, biomembrane lysis buffer was added; this buffer was used to break down the cell and nuclear membranes, releasing genomic DNA from the cells. Anhydrous ethanol was then added to specifically adsorb the DNA onto the adsorption column. Finally, ddH₂O was used to remove impurities such as proteins and RNA, and the purified DNA was eluted using a mixture of 80% formamide and 5% EDTA (volume ratio 2:1). This yielded the pre-treated blood sample ready for testing.

[0049] 2. PCR amplification Add 1 μL of pretreated blood sample to the PCR system for amplification, and obtain the amplified PCR product. The primer pair in the PCR system consists of a 100 μM upstream primer and a 100 μM downstream primer, with the nucleotide sequence of the upstream primer being SEQ ID NO.2 and the nucleotide sequence of the downstream primer being SEQ ID NO.3. The parameters for the amplification process can be set as follows: initialization temperature can be 95℃, and time can be 5 min. The number of cycles can be 45. During the cycles, the denaturation phase temperature can be 95℃, and time can be 30 s; the annealing phase temperature can be 55℃, and time can be 50 s; the extension phase temperature can be 72℃, and time can be 45 s; and the final extension phase temperature after cycling can be 72℃, and time can be 10 min. For specific amplification procedures, refer to existing technologies.

[0050] 3. Probe binding Add 20 μL of PCR product to 0.4 μL of oligonucleotide probe, incubate at 95 °C for 5 min to anneal and bind, and then obtain the sample.

[0051] 4. Sample detection based on SERS technology Take 5 μL of the sample obtained in step 3 and drop it onto the prepared glass slide with silver nanoparticles, then air dry it at room temperature. After the sample is dry, use a small Raman spectrometer (model NR785E10W-Plus) to perform SRES detection at an excitation wavelength of 785 nm, a detection power of 30 mW, and an integration time of 500 ms to obtain the SRES spectrum of the blood sample.

[0052] Example 6 Patient Zhang X, female, 70 years old, was diagnosed with breast cancer with a BRCA2 gene mutation in July 2025. Peripheral blood was collected from the patient, and a probe was used to detect the BRCA2 gene mutation, including the following steps: 1. Collect blood samples from the patient. 100 μL of peripheral blood was collected from the patient, and cell lysis buffer was added to lyse the cells. Then, biomembrane lysis buffer was added; this buffer was used to break down the cell and nuclear membranes, releasing genomic DNA from the cells. Anhydrous ethanol was then added to specifically adsorb the DNA onto the adsorption column. Finally, ddH₂O was used to remove impurities such as proteins and RNA, and the purified DNA was eluted using a mixture of 80% formamide and 5% EDTA (volume ratio 2:1). This yielded the pre-treated blood sample ready for testing.

[0053] 2. PCR amplification Add 1 μL of pretreated blood sample to the PCR system for amplification, and obtain the amplified PCR product. The primer pair in the PCR system consists of a 100 μM upstream primer and a 100 μM downstream primer, with the nucleotide sequence of the upstream primer being SEQ ID NO.2 and the nucleotide sequence of the downstream primer being SEQ ID NO.3. The parameters for the amplification process can be set as follows: initialization temperature can be 95℃, and time can be 5 min. The number of cycles can be 45. During the cycles, the denaturation phase temperature can be 95℃, and time can be 30 s; the annealing phase temperature can be 55℃, and time can be 50 s; the extension phase temperature can be 72℃, and time can be 45 s; and the final extension phase temperature after cycling can be 72℃, and time can be 10 min. For specific amplification procedures, refer to existing technologies.

[0054] 3. Probe binding Add 20 μL of PCR product to 0.4 μL of oligonucleotide probe, incubate at 95 °C for 5 min to anneal and bind, and then obtain the sample.

[0055] 4. Sample detection based on SERS technology Take 5 μL of the sample obtained in step 3 and drop it onto the prepared glass slide with silver nanoparticles, then air dry it at room temperature. After the sample is dry, use a small Raman spectrometer (model NR785E10W-Plus) to perform SRES detection at an excitation wavelength of 785 nm, a detection power of 30 mW, and an integration time of 500 ms to obtain the SRES spectrum of the blood sample.

[0056] Example 7 Patient Li XX, female, 55 years old, was diagnosed with breast cancer in February 2025, but did not carry a BRCA2 gene mutation. Peripheral blood was collected from the patient, and a probe was used to detect BRCA2 gene mutations, including the following steps: 1. Collect blood samples from the patient. 100 μL of peripheral blood was collected from the patient, and cell lysis buffer was added to lyse the cells. Then, biomembrane lysis buffer was added; this buffer was used to break down the cell and nuclear membranes, releasing genomic DNA from the cells. Anhydrous ethanol was then added to specifically adsorb the DNA onto the adsorption column. Finally, ddH₂O was used to remove impurities such as proteins and RNA, and the purified DNA was eluted using a mixture of 80% formamide and 5% EDTA (volume ratio 2:1). This yielded the pre-treated blood sample ready for testing.

[0057] 2. PCR amplification Add 1 μL of pretreated blood sample to the PCR system for amplification, and obtain the amplified PCR product. The primer pair in the PCR system consists of a 100 μM upstream primer and a 100 μM downstream primer, with the nucleotide sequence of the upstream primer being SEQ ID NO.2 and the nucleotide sequence of the downstream primer being SEQ ID NO.3. The parameters for the amplification process can be set as follows: initialization temperature can be 95℃, and time can be 5 min. The number of cycles can be 45. During the cycles, the denaturation phase temperature can be 95℃, and time can be 30 s; the annealing phase temperature can be 55℃, and time can be 50 s; the extension phase temperature can be 72℃, and time can be 45 s; and the final extension phase temperature after cycling can be 72℃, and time can be 10 min. For specific amplification procedures, refer to existing technologies.

[0058] 3. Probe binding Add 20 μL of PCR product to 0.4 μL of oligonucleotide probe, incubate at 95 °C for 5 min to anneal and bind, and then obtain the sample.

[0059] 4. Sample detection based on SERS technology Take 5 μL of the sample obtained in step 3 and drop it onto the prepared glass slide with silver nanoparticles, then air dry it at room temperature. After the sample is dry, use a small Raman spectrometer (model NR785E10W-Plus) to perform SRES detection at an excitation wavelength of 785 nm, a detection power of 30 mW, and an integration time of 500 ms to obtain the SRES spectrum of the blood sample.

[0060] In addition, blood samples from three patients in Examples 5 to 7 were used to detect BRCA2 gene mutations using existing NGS sequencing methods, and these samples served as a control group. Since NGS sequencing is a prior art technology, this detection process will not be described in detail in this protocol.

[0061] Table 1 summarizes the BRCA2 gene mutation results obtained from blood samples of the three patients in Examples 5 to 7 using both NGS sequencing and the detection method described in this protocol. Table 1 shows the BRCA2 gene mutation detection results obtained from peripheral blood samples of the three patients in Examples 5 to 7 using both NGS sequencing and the detection method described in this protocol.

[0062] Analysis of the table above shows that the detection results of BRCA2 gene mutations by NGS sequencing and the detection method of this scheme in Examples 5 to 7 of this scheme are consistent. Therefore, it can be shown that the detection method provided by this scheme can accurately and effectively detect whether BRCA2 gene mutations have occurred.

[0063] Furthermore, this scheme also summarizes the three sets of SERS spectra obtained in Examples 5 to 7 into a single figure, and the results are as follows: Figure 2 As shown. By analyzing... Figure 2 Analysis showed that the blood samples from patients in Examples 5 and 6, after being tested using the detection method provided in this protocol, had a concentration of 1188 cm⁻¹. -1 and 1393cm -1 Characteristic peaks appeared at all locations, indicating that the two patients in Examples 5 and 6 had BRCA2 gene mutations. This result was consistent with the results of NGS sequencing. The blood sample from the patient in Example 7, after being tested using the method provided in this protocol, showed a peak at 1188 cm⁻¹. -1 and 1393cm -1 No characteristic peaks were observed at any of the locations, and this detection result was consistent with that of NGS sequencing. Therefore, the verification experiments in Examples 5 and 7 demonstrate that the detection method provided in this scheme can accurately detect BRCA2 gene mutations.

[0064] The basic principles, main features, and advantages of this invention have been described above. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made without departing from the spirit and scope of the invention, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection claimed by this invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting BRCA2 gene mutations, characterized in that, It includes: The blood sample to be tested was amplified by PCR using a primer pair with the nucleotide sequence of SEQ ID NO.2 for the upstream primer and the nucleotide sequence of SEQ ID NO.3 for the downstream primer. The amplified PCR product was co-incubated with an oligonucleotide probe with the nucleotide sequence SEQ ID NO.1 and a cy3 fluorescent group attached to its 5' end to obtain a sample; The sample was detected at 1188 cm⁻¹ using SERS technology. -1 and 1393cm -1 Does the sample contain a characteristic peak at 1188 cm⁻¹? Make the following judgment: -1 and 1393cm -1 If characteristic peaks are present at all locations, it indicates that the blood sample to be tested contains a BRCA2 gene mutation; otherwise, it indicates that the blood sample to be tested does not contain a BRCA2 gene mutation.

2. The method for detecting BRCA2 gene mutations as described in claim 1, characterized in that, The blood sample to be tested needs to undergo the following pretreatment before PCR amplification: Blood samples were obtained, and cell lysis buffer and biomembrane lysis buffer were added sequentially to lyse the peripheral blood samples. After lysis, DNA adsorption buffer was added, and the samples were washed with detergent to obtain the blood samples to be tested.

3. The method for detecting BRCA2 gene mutations as described in claim 2, characterized in that, The cell lysis buffer was prepared by mixing protein lysin K and PBS at a volume ratio of 1:

9. And / or, the biofilm lysis buffer is a Lysis Buffer; And / or, the DNA adsorption solution is anhydrous ethanol; And / or, the detergent comprises ddH2O and a washing buffer; the washing buffer is a mixture of 80% formamide and 5% EDTA at a volume ratio of 2:

1.

4. The method for detecting BRCA2 gene mutations as described in claim 3, characterized in that, The washing agent is used to wash the blood sample after adding the DNA adsorption solution by washing it multiple times with ddH2O; then, a washing buffer with a volume ratio of 5-6:25 to ddH2O is added to the washed blood sample for further washing, thereby obtaining the pre-treated blood sample.

5. The method for detecting BRCA2 gene mutations as described in claim 3, characterized in that, In the pretreatment of the blood samples, the volume ratio of cell lysis buffer to blood sample and the volume ratio of biological lysis buffer to blood sample are both 1:2; the volume ratio of DNA adsorption buffer to blood sample is 1:10 to 1:

20.

6. The method for detecting BRCA2 gene mutations as described in claim 1, characterized in that, The SERS technology uses a Raman spectrometer for detection. Before detection, the sample needs to be dropped onto the substrate and dried. The excitation wavelength of the Raman spectrometer is 780nm-790nm, the excitation power is 25mW-30mW, and the integration time is 500ms. And / or, the concentration of the oligonucleotide probe is 8 μM; And / or, the volume ratio of the amplified PCR product to the oligonucleotide probe during co-incubation is 50:

1.

7. The method for detecting BRCA2 gene mutations as described in claim 6, characterized in that, The substrate is a silver nanoparticle substrate; the preparation method of the silver nanoparticle substrate is as follows: the spherical silver nanoparticle-citric acid suspension is centrifuged at 6500 r / min to precipitate, and the supernatant is removed; ddH2O is added to the precipitated spherical silver nanoparticles to make up to 80 times the volume of the spherical silver nanoparticles before centrifugation to obtain a second mixture; 5 μL of the second mixture is dropped onto a silicon wafer and dried at room temperature to obtain the silver nanoparticle substrate.

8. A system for detecting BRCA2 gene mutations, characterized in that, It employs the method for detecting BRCA2 gene mutations as described in any one of claims 1-7; the system comprises: The blood processing module is used to sequentially pre-process, amplify, co-incubate, and dry the blood sample to be tested, thereby obtaining the sample to be tested. A Raman spectrometer is used to acquire molecular vibrational / rotational information of a sample to be tested and to generate a Raman spectrum. The data processing module has a built-in 1188cm -1 and 1393cm -1 The dual-peak identification algorithm; the data processing module is used to acquire Raman spectra and make the following judgment: if the Raman spectrum of the sample is at 1188 cm⁻¹... -1 and 1393cm -1 If a characteristic peak is present at all locations, it indicates that the blood sample to be tested has a BRCA2 gene mutation and outputs "mutation positive"; otherwise, it indicates that the blood sample to be tested does not have a BRCA2 gene mutation and outputs "mutation negative".

9. A reagent kit, characterized in that, It is used to store the pre-processed blood sample to be tested in the method for detecting BRCA2 gene mutations as described in any one of claims 2-5.

10. A probe, characterized in that, It is an oligonucleotide probe used in the method for detecting BRCA2 gene mutations as described in any one of claims 1-7; the probe is used to bind to the BRCA2 gene sequence when the BRCA2 gene is mutated to capture the BRCA2 gene.

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