SERS (Surface Enhanced Raman Scattering) detection method for thyroid cancer BRAF V600E mutation

Abstract

The invention provides an SERS (Surface Enhanced Raman Scattering) detection method for thyroid cancer BRAF V600E mutation, and belongs to the technical field of detection. A probe is composed of oligonucleotide of which the 5'end is connected with a cy3 fluorophore. During detection, a tissue sample is subjected to splitting decomposition and PCR amplification and then is hybridized with the probe, and uncombined probes are removed by adopting a wash buffer containing 95% formamide and 5% EDTA (Ethylene Diamine Tetraacetic Acid) and a DNA purification kit. A purified product is dropwise added to a KI aptamer modified silver nanoparticle coffee ring substrate, and an SERS spectrum is collected through 785 nm laser excitation. Mutation is judged through cy3 characteristic double peaks at 1188 cm <-1 > and 1393 cm <-1 >, and detection is completed within 4 hours. Clinical verifications show that the consistency rate with a gene detection result is 100%, the sensitivity reaches 0.1% mutation frequency, the limitations of long consumed time and high cost of traditional PCR / NGS are overcome, and the kit is suitable for intraoperative rapid diagnosis and targeted treatment guidance.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a SERS detection method for BRAF V600E mutations in thyroid cancer. Background Technology

[0002] BRAF is a proto-oncogene located on chromosome 7 (7q34) that encodes a serine / threonine protein kinase and is a core member of the RAS-RAF-MEK-ERK signaling pathway (i.e., the MAPK pathway). This pathway regulates key physiological processes such as cell proliferation, differentiation, and survival. Under normal circumstances, it is strictly regulated by upstream signals to ensure orderly cellular activity. V600E is the most common pathogenic mutation type of the BRAF gene, named after a change in the gene sequence: the 600th amino acid is mutated from valine (V) to glutamate (E). This mutation leads to an abnormal BRAF protein structure, causing it to escape upstream signal regulation and remain in an activated state. This overactivation of the MAPK pathway results in uncontrolled cell proliferation and abnormal differentiation, ultimately inducing tumors.

[0003] Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer (accounting for 70%-80% of all thyroid cancers). The BRAF V600E mutation has the highest incidence in PTC, ranging from 40% to 80% in different studies, and is one of the major driver mutations in PTC. In highly malignant undifferentiated thyroid carcinoma (ATC), the BRAF V600E mutation is detectable in approximately 20%-40%, often indicating rapid tumor progression and a very poor prognosis. It occurs less frequently in a small number of follicular thyroid carcinomas (FTC) (approximately 5%-10%), but a positive mutation may indicate a more aggressive biological behavior.

[0004] Determining the presence of the BRAF V600E mutation can play a role in three aspects: auxiliary diagnosis, prognostic assessment, and treatment guidance. In diagnosis, for thyroid nodules suspected of malignancy by ultrasound or with inconclusive results from fine-needle aspiration biopsy, a positive mutation indicates a malignancy probability exceeding 95%, significantly improving diagnostic accuracy. While a negative mutation does not completely rule out malignancy, it reduces the risk. In prognostic assessment, a positive mutation typically indicates a worse clinical outcome, including a higher rate of lymph node metastasis, risk of distant metastasis, recurrence rate, decreased sensitivity to radioactive iodine therapy, and shortened overall survival. In treatment decisions, the mutation status can guide the selection of surgical scope (e.g., more aggressive total thyroidectomy with lymph node dissection), closer postoperative follow-up monitoring, and for recurrent, metastatic, or iodine-refractory cases, targeted therapy combining BRAF inhibitors and MEK inhibitors can effectively shrink tumors and prolong progression-free survival, providing important treatment options for these patients.

[0005] Currently, the main clinical methods for detecting BRAF V600E mutations include real-time quantitative PCR (qPCR), digital PCR (dPCR), and next-generation sequencing (NGS). qPCR relies on the specificity of primer and probe design, making it susceptible to inhibitors or cross-reactivity in the sample, potentially leading to false positives. Digital PCR is significantly more expensive than qPCR, has a limited sample size that can be detected in a single reaction, and requires advanced operational techniques. Next-generation sequencing (NGS) is also expensive and has a longer processing time. All these methods primarily use fine-needle aspiration thyroid cell specimens or surgically removed tumor tissue as the detection sample, necessitating a high proportion of tumor cells in the sample to avoid false negatives. Therefore, developing a simple, rapid, sensitive, and cost-effective novel monitoring method is of great significance for the rapid detection of BRAF V600E mutations in clinical practice.

[0006] In recent years, surface-enhanced Raman spectroscopy (SERS) has developed rapidly, and an increasing number of studies are using SERS for the detection of tissues and biomolecules in vivo. Therefore, developing a new gene mutation detection method using the technological advantages of SERS has high clinical application prospects. Summary of the Invention

[0007] The purpose of this invention is to provide a SERS method for detecting BRAF V600E mutations in thyroid cancer. This invention utilizes SERS technology to detect BRAF V600E mutations in thyroid cancer. Specifically, a probe linked to a fluorescent group specifically binds to the BRAF V600E mutation. Loosely bound probes are removed by washing with a purification kit and wash buffer. The peak generated by the fluorescent group in the final SERS product is detected, enhancing the detection capability of the SERS substrate for BRAF V600E gene mutations and improving the sensitivity and accuracy of SERS for BRAF V600E gene mutation detection. This method features high sensitivity, rapid detection, and low cost.

[0008] This application first pre-treats the test sample by adding lysis buffer and heating in a metal bath to release the sample DNA. The sample DNA is then used to prepare a PCR system to amplify the BRAF gene fragment. The amplified gene is then incubated with a probe linked to a reporter molecule. Unbound probes are washed away using a wash buffer and a PCR product purification kit. The purified product is then dropped onto a coffee ring made from baked silver particles and dried before being examined on a slide. This method can complete the detection of BRAF V600E mutations in a patient's thyroid gland in just four hours.

[0009] To solve the above-mentioned technical problems, the specific technical solution of the present invention is as follows:

[0010] A method for preparing a probe to detect BRAF V600E mutations in thyroid cancer, comprising the following steps:

[0011] An oligonucleotide probe with a 5' end linked to a cy3 fluorescent group was synthesized, and its nucleotide sequence is cta gct aca gagaaa tct cga, as shown in SEQ ID NO:1;

[0012] The probe concentration was adjusted to 10 μM and stored in a light-protected environment at -20℃.

[0013] A SERS detection method for BRAF V600E mutation in thyroid cancer includes the following steps:

[0014] Take tissue samples, add 50-100 μL of Lysis Buffer, and heat in a 95°C metal bath for 20 min to obtain lysis products;

[0015] Add 1 μL of the lysis product to the PCR system, containing 100 μM of the upstream primer att tct tca tga aga cct cacagt as shown in SEQ ID NO:2, and 100 μM of the downstream primer gat cca gac aac tgt tca aac tga as shown in SEQ ID NO:3. The amplification program is: 95℃ for 5 min; 45× [95℃ for 30 s, 55℃ for 50 s, 72℃ for 45 s]; 72℃ for 10 min.

[0016] Add 0.4 μL of the probe to 20 μL of PCR product and incubate at 95 °C for 5 min for annealing and binding;

[0017] The product was purified using a DNA purification kit and a wash buffer containing 95% formamide and 5% EDTA.

[0018] The purified sample was dropped onto a silver nanoparticle substrate and dried at 45°C.

[0019] SERS spectra were detected using 785nm laser excitation, 30mW power, and an integration time of 500ms, and passed through a 1188cm⁻¹ laser. -1 and 1393cm -1 The mutation was determined by the characteristic peak of cy3.

[0020] In the above technical solution, the wash buffer purification includes three steps:

[0021] a) Mix the probe binding product with an equal volume of DNA purification column solution I and incubate at room temperature for 1 min;

[0022] b) Add 40 μL wash buffer, incubate at 65°C for 5 min, centrifuge at 16,000 g and discard the liquid. Repeat three times.

[0023] c) Wash with 700 μL and 500 μL of solution II in sequence, centrifuge at 16,000 g, and then elute with 50 μL of solution III.

[0024] In the above technical solution, the silver nanoparticle substrate is prepared through the following steps:

[0025] Take 6×10 8 Spherical silver nanoparticles / mL solution of sodium citrate were centrifuged at 7500 rpm for 10 min.

[0026] Add an equal volume of KI aptamer to the precipitate, let stand for 1 hour, and then dilute to 20 times the volume with ddH2O.

[0027] Add 5 μL of solution to a silicon wafer and dry at 45°C to form a coffee ring structure.

[0028] In the above technical solution, the PCR amplification needs to be verified by 1.5% agarose gel electrophoresis under the conditions of 120V voltage for 40min.

[0029] A BRAF V600E mutation detection kit, comprising:

[0030] The cy3-labeled probe has the nucleotide sequence cta gct aca gag aaa tct cga, as shown in SEQ ID NO:1;

[0031] Specific primer pairs: upstream att tct tca tga aga cct cac agt as shown in SEQ ID NO:2, downstream gat cca gac aac tgt tca aac tga as shown in SEQ ID NO:3;

[0032] A wash buffer solution containing 95% formamide and 5% EDTA;

[0033] KI aptamer-modified silver nanoparticle substrate.

[0034] In the above technical solution, the kit further includes Lysis Buffer lysis buffer, DNA purification column and solutions I / II / III.

[0035] A BRAF V600E mutation interpretation system, integrating:

[0036] Raman spectrometer, with an excitation wavelength of 785±5nm and a power of 30±5mW;

[0037] Data processing module, built-in 1188cm -1 and 1393cm -1 Bimodal peak identification algorithm;

[0038] The output interface displays a "mutation positive" or "mutation negative" conclusion.

[0039] A prognostic assessment method for thyroid cancer is proposed, which uses the method to detect BRAF V600E mutation in patient tissue samples; a positive mutation indicates the need to expand the surgical scope and initiate targeted therapy, while a negative mutation indicates the use of a conventional surgical procedure.

[0040] An application of a BRAF V600E mutation probe is described, in which the probe is used to prepare a recurrence monitoring reagent for thyroid cancer after surgery, and the treatment plan is adjusted by periodically detecting the mutation status in circulating tumor DNA.

[0041] Beneficial effects:

[0042] This invention optimizes the purification effect of the reagent kit by using wash buffer treatment, washing away probes that do not bind well and reducing the error of positive test results.

[0043] This invention constructs a probe that binds to a fluorescent reporter molecule and attaches it to a mutation site. The presence or absence of a characteristic peak in the fluorescent molecule is used to determine the existence of a mutation, reducing the difficulty of identifying mutations through subtle changes in characteristic peaks. The entire detection process takes only 4 hours. It is suitable for rapid detection of BRAF V600E mutations. Attached Figure Description

[0044] Figure 1 The probe successfully detected the SERS spectrum of the BRAF V600E mutation. Detailed Implementation

[0045] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. However, the following embodiments are only for explaining the present invention, and the scope of protection of the present invention should include all the contents of the claims. Moreover, through the description of the following embodiments, those skilled in the art can fully implement all the contents of the claims of the present invention.

[0046] Example 1:

[0047] Patient Yang X, male, 62 years old, was pathologically diagnosed with BRAF V600E mutation in June 2025. Frozen pathological tissue was collected from the patient, and a probe was used to detect BRAF V600E mutation in thyroid cancer, including the following steps:

[0048] 1. DNA extraction from patient tissue samples:

[0049] Take a patient tissue sample and add 50-100 μL of Lysis Buffer to cover the tissue sample. Incubate in a 95°C metal bath for 20 min to obtain lysis products.

[0050] 2. BRAF gene fragment amplification and probe binding:

[0051] (1) Take 1 μL of lysis product, add 12.5 μL Teq Mix, 1 μL BRAF upstream primer, 1 μL BRAF downstream primer, and 9.5 μL ddH2O to make a 25 μL system. PCR amplify the BRAF gene at 95℃ for 5 min, for 45 cycles (95℃ for 30 s, 55℃ for 50 s, 72℃ for 45 s), at 72℃ for 10 min, and incubate at 4℃.

[0052] (2) Take 5 μL of the 25 μL PCR product and verify the amplification results by 1.5% agarose gel electrophoresis.

[0053] (3) Add 0.4 μL of BRAF probe with a concentration of 10 μM to 20 μL of PCR product, heat at 95℃ for 5 min, and anneal.

[0054] 3. Sample purification and elution:

[0055] (1) Mix 20 μL of the PCR product after binding the probe with an equal volume of solution I in the DNA purification kit, add it to the DNA purification column, and let it stand at room temperature for 1 minute.

[0056] (2) Add 40 μL of wash buffer to the DNA purification column and heat in a water bath at 65°C for 5 min.

[0057] (3) Centrifuge at the highest speed of 16,000g for 1 minute and discard the liquid in the collection tube.

[0058] (4) Repeat steps (2) and (3) twice.

[0059] (5) Add 700 μL of Solution II to the DNA purification column and let it stand at room temperature for 1 minute.

[0060] (6) Centrifuge at the highest speed for 1 minute to wash away impurities. Discard the liquid in the collection tube.

[0061] (7) Add another 500 μL of solution II, centrifuge at the highest speed for 1 minute to further wash away impurities. Discard the liquid in the collection tube.

[0062] (8) Centrifuge at the highest speed for 1 minute to remove residual liquid and allow residual ethanol to evaporate fully.

[0063] (9) Place the DNA purification column on a 1.5 mL centrifuge tube, add 50 μL of Solution III to the column surface, and let stand for 1 minute. Use a 1.5 mL centrifuge tube as the collection tube.

[0064] (10) Centrifuge at the highest speed for 1 minute, and the resulting liquid is high-purity DNA.

[0065] 4. SERS detection and data output:

[0066] (1) Take a concentration of 6×10 8 A solution of spherical silver nanoparticles / mL and sodium citrate was centrifuged at 7500 rpm for 10 min at room temperature. The supernatant was discarded, and an equal volume of KI aptamer was added to the precipitate. After standing for 1 h, ddH2O was added to bring the volume up to 20 times the volume of the silver nanoparticles.

[0067] (2) Take 5 μL of the above solution and drop it onto the silicon wafer. After drying in an environment of 45°C, a DNA nanoparticle chip is formed on the silicon wafer.

[0068] (3) Take 5 μL of the purified sample and drop it onto the DNA nanoparticle chip. Place the silicon wafer with the tissue disruption solution added to it back into the environment at 45°C to dry.

[0069] (4) After the sample is dried, SERS detection is performed using a small Raman spectrometer (NR785E10W-Plus). The excitation wavelength of the Raman spectrometer is about 785nm, the detection power is 30mW, and the integration time is 500ms to obtain the SERS spectrum of the blood sample.

[0070] (5) such as Figure 1 As shown in Table 1, the characteristic peak of cy3 (1188 cm⁻¹) in patient tissue specimens detected in SERS spectra after combining with the probe was observed. -1 ) and (1393cm -1 The comparison revealed a characteristic peak with a BRAF V600E site mutation, consistent with the patient's gene testing results.

[0071] Example 2:

[0072] Patient Sun X, female, 45 years old, was pathologically diagnosed with papillary thyroid carcinoma in June 2025, without BRAF V600E mutation. Frozen pathological tissue was collected from the patient, and BRAF V600E mutation was detected in the thyroid cancer using a probe, including the following steps:

[0073] 1. DNA extraction from patient tissue samples:

[0074] Take a patient tissue sample and add 50-100 μL of Lysis Buffer to cover the tissue sample. Incubate in a 95°C metal bath for 20 min to obtain lysis products.

[0075] 2. BRAF gene fragment amplification and probe binding:

[0076] (1) Take 1 μL of lysis product, add 12.5 μL Teq Mix, 1 μL BRAF upstream primer, 1 μL BRAF downstream primer, and 9.5 μL ddH2O to make a 25 μL system. PCR amplify the BRAF gene at 95℃ for 5 min, for 45 cycles (95℃ for 30 s, 55℃ for 50 s, 72℃ for 45 s), at 72℃ for 10 min, and incubate at 4℃.

[0077] (2) Take 5 μL of the 25 μL PCR product and verify the amplification results by 1.5% agarose gel electrophoresis.

[0078] (3) Add 0.4 μL of BRAF probe with a concentration of 10 μM to 20 μL of PCR product, heat at 95℃ for 5 min, and anneal.

[0079] 3. Sample purification and elution:

[0080] (1) Mix 20 μL of the PCR product after binding the probe with an equal volume of solution I in the DNA purification kit, add it to the DNA purification column, and let it stand at room temperature for 1 minute.

[0081] (2) Add 40 μL of wash buffer to the DNA purification column and heat in a water bath at 65°C for 5 min.

[0082] (3) Centrifuge at the highest speed of 16,000g for 1 minute and discard the liquid in the collection tube.

[0083] (4) Repeat steps (2) and (3) twice.

[0084] (5) Add 700 μL of Solution II to the DNA purification column and let it stand at room temperature for 1 minute.

[0085] (6) Centrifuge at the highest speed for 1 minute to wash away impurities. Discard the liquid in the collection tube.

[0086] (7) Add another 500 μL of solution II, centrifuge at the highest speed for 1 minute to further wash away impurities. Discard the liquid in the collection tube.

[0087] (8) Centrifuge at the highest speed for 1 minute to remove residual liquid and allow residual ethanol to evaporate fully.

[0088] (9) Place the DNA purification column on a 1.5 mL centrifuge tube, add 50 μL of Solution III to the column surface, and let stand for 1 minute. Use a 1.5 mL centrifuge tube as the collection tube.

[0089] (10) Centrifuge at the highest speed for 1 minute, and the resulting liquid is high-purity DNA.

[0090] 4. SERS detection and data output:

[0091] (1) Take a concentration of 6×10 8 A solution of spherical silver nanoparticles / mL and sodium citrate was centrifuged at 7500 rpm for 10 min at room temperature. The supernatant was discarded, and an equal volume of KI aptamer was added to the precipitate. After standing for 1 h, ddH2O was added to bring the volume up to 20 times the volume of the silver nanoparticles.

[0092] (2) Take 5 μL of the above solution and drop it onto the silicon wafer. After drying in an environment of 45°C, a DNA nanoparticle chip is formed on the silicon wafer.

[0093] (3) Take 5 μL of the purified sample and drop it onto the DNA nanoparticle chip. Place the silicon wafer with the tissue disruption solution added to it back into the environment at 45°C to dry.

[0094] (4) After the sample is dried, SERS detection is performed using a small Raman spectrometer (NR785E10W-Plus). The excitation wavelength of the Raman spectrometer is about 785nm, the detection power is 30mW, and the integration time is 500ms to obtain the SERS spectrum of the blood sample.

[0095] (5) such as Figure 1 As shown in Table 1, the characteristic peak of cy3 (1188 cm⁻¹) in patient tissue specimens detected in SERS spectra after combining with the probe was observed. -1 ) and (1393cm -1 Compared with other groups, no characteristic peak was observed at the BRAF V600E site, indicating no BRAF V600E mutation.

[0096] Example 3:

[0097] Patient Zhang X, male, 56 years old, was diagnosed with papillary thyroid carcinoma in July 2025, without BRAF V600E mutation. Frozen pathological tissue was collected from the patient, and a probe was used to detect BRAF V600E mutation in the thyroid cancer, including the following steps:

[0098] 1. DNA extraction from patient tissue samples:

[0099] Take a patient tissue sample and add 50-100 μL of Lysis Buffer to cover the tissue sample. Incubate in a 95°C metal bath for 20 min to obtain lysis products.

[0100] 2. BRAF gene fragment amplification and probe binding:

[0101] (1) Take 1 μL of lysis product, add 12.5 μL Teq Mix, 1 μL BRAF upstream primer, 1 μL BRAF downstream primer, and 9.5 μL ddH2O to make a 25 μL system. PCR amplify the BRAF gene at 95℃ for 5 min, for 45 cycles (95℃ for 30 s, 55℃ for 50 s, 72℃ for 45 s), at 72℃ for 10 min, and incubate at 4℃.

[0102] (2) Take 5 μL of the 25 μL PCR product and verify the amplification results by 1.5% agarose gel electrophoresis.

[0103] (3) Add 0.4 μL of BRAF probe with a concentration of 10 μM to 20 μL of PCR product, heat at 95℃ for 5 min, and anneal.

[0104] 3. Sample purification and elution:

[0105] (1) Mix 20 μL of the PCR product after binding the probe with an equal volume of solution I in the DNA purification kit, add it to the DNA purification column, and let it stand at room temperature for 1 minute.

[0106] (2) Add 40 μL of wash buffer to the DNA purification column and heat in a water bath at 65°C for 5 min.

[0107] (3) Centrifuge at the highest speed of 16,000g for 1 minute and discard the liquid in the collection tube.

[0108] (4) Repeat steps (2) and (3) twice.

[0109] (5) Add 700 μL of Solution II to the DNA purification column and let it stand at room temperature for 1 minute.

[0110] (6) Centrifuge at the highest speed for 1 minute to wash away impurities. Discard the liquid in the collection tube.

[0111] (7) Add another 500 μL of solution II, centrifuge at the highest speed for 1 minute to further wash away impurities. Discard the liquid in the collection tube.

[0112] (8) Centrifuge at the highest speed for 1 minute to remove residual liquid and allow residual ethanol to evaporate fully.

[0113] (9) Place the DNA purification column on a 1.5 mL centrifuge tube, add 50 μL of Solution III to the column surface, and let stand for 1 minute. Use a 1.5 mL centrifuge tube as the collection tube.

[0114] (10) Centrifuge at the highest speed for 1 minute, and the resulting liquid is high-purity DNA.

[0115] 4. SERS detection and data output:

[0116] (1) Take a concentration of 6×10 8 A solution of spherical silver nanoparticles / mL and sodium citrate was centrifuged at 7500 rpm for 10 min at room temperature. The supernatant was discarded, and an equal volume of KI aptamer was added to the precipitate. After standing for 1 h, ddH2O was added to bring the volume up to 20 times the volume of the silver nanoparticles.

[0117] (2) Take 5 μL of the above solution and drop it onto the silicon wafer. After drying in an environment of 45°C, a DNA nanoparticle chip is formed on the silicon wafer.

[0118] (3) Take 5 μL of the purified sample and drop it onto the DNA nanoparticle chip. Place the silicon wafer with the tissue disruption solution added to it back into the environment at 45°C to dry.

[0119] (4) After the sample is dried, SERS detection is performed using a small Raman spectrometer (NR785E10W-Plus). The excitation wavelength of the Raman spectrometer is about 785nm, the detection power is 30mW, and the integration time is 500ms to obtain the SERS spectrum of the blood sample.

[0120] (5) such as Figure 1 As shown in Table 1, the characteristic peak of cy3 (1188 cm⁻¹) in patient tissue specimens detected in SERS spectra after combining with the probe was observed. -1 ) and (1393cm -1 Compared with other groups, no characteristic peak was observed at the BRAF V600E site, which was consistent with the patient's gene testing results.

[0121] Analysis of test and experimental results:

[0122] like Figure 1 As shown, Figure 1 To ensure successful detection of the BRAF V600E mutation by the probe in the SERS spectrum, the experiment used the probe to detect patient tissues with and without the BRAF V600E mutation. Combining the results of patient SERS and gene testing for BRAF V600E mutations in Table 1 below, it can be concluded that in Example 1, the cy3 characteristic peak (1188 cm⁻¹) was detected in the patient tissue samples in the SERS spectrum after combining with the probe. -1 ) and (1393cm -1 The comparison revealed a characteristic peak with a BRAF V600E mutation, consistent with the patient's gene testing results; the characteristic peak of cy3 (1188 cm⁻¹) in the SERS spectra of patient tissue specimens after probe binding was detected in Examples 2 and 3. -1 ) and (1393cm -1 Compared with other groups, no characteristic peak was observed at the BRAF V600E site, which was consistent with the patient's gene testing results.

[0123] Table 1. Results of SERS and BRAF V600E mutation detection in patients.

[0124] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing a probe to detect BRAF V600E mutations in thyroid cancer, characterized in that, Includes the following steps: An oligonucleotide probe with a 5' end linked to a cy3 fluorescent group was synthesized, and its nucleotide sequence is cta gct aca gag aaatct cga, as shown in SEQ ID NO:1; The probe concentration was adjusted to 10 μM and stored in a light-protected environment at -20℃.

2. A SERS detection method for BRAF V600E mutation in thyroid cancer, characterized in that, Includes the following steps: Take tissue samples, add 50-100 μL of Lysis Buffer, and heat in a 95°C metal bath for 20 min to obtain lysis products; Add 1 μL of the lysis product to the PCR system, containing 100 μM of the upstream primer att tct tca tga aga cct cac agt as shown in SEQ ID NO:2 and 100 μM of the downstream primer gat cca gac aac tgt tca aac tga as shown in SEQ ID NO:

3. The amplification program is as follows: 95℃ for 5 min; 45× [95℃ for 30 s, 55℃ for 50 s, 72℃ for 45 s]; 72℃ for 10 min. Take 20 μL of PCR product and add 0.4 μL of the probe described in claim 1, incubate at 95 °C for 5 min for annealing and binding; The product was purified using a DNA purification kit and a wash buffer containing 95% formamide and 5% EDTA. The purified sample was dropped onto a silver nanoparticle substrate and dried at 45°C. SERS spectra were detected using 785nm laser excitation, 30mW power, and an integration time of 500ms, and passed through a 1188cm⁻¹ laser. -1 and 1393cm -1 The mutation was determined by the characteristic peak of cy3.

3. The method according to claim 2, characterized in that, The wash buffer purification process involves three steps: a) Mix the probe binding product with an equal volume of DNA purification column solution I and incubate at room temperature for 1 min; b) Add 40 μL wash buffer, incubate at 65°C for 5 min, centrifuge at 16,000 g and discard the liquid. Repeat three times. c) Wash with 700 μL and 500 μL of solution II in sequence, centrifuge at 16,000 g, and then elute with 50 μL of solution III.

4. The method according to claim 2, characterized in that, The silver nanoparticle substrate is prepared by the following steps: Take 6×10 8 Spherical silver nanoparticles / mL solution of sodium citrate were centrifuged at 7500 rpm for 10 min. Add an equal volume of KI aptamer to the precipitate, let stand for 1 hour, and then dilute to 20 times the volume with ddH2O. Add 5 μL of solution to a silicon wafer and dry at 45°C to form a coffee ring structure.

5. The method according to claim 2, characterized in that, After PCR amplification, the results need to be verified by 1.5% agarose gel electrophoresis at 120V for 40 minutes.

6. A BRAF V600E mutation detection kit, characterized in that, Include: The oligonucleotide probe of claim 1; Specific primer pairs: upstream att tct tca tga aga cct cac agt as shown in SEQ ID NO:2, downstream gatcca gac aac tgt tca aac tga as shown in SEQ ID NO:3; A wash buffer solution containing 95% formamide and 5% EDTA; KI aptamer-modified silver nanoparticle substrate.

7. The reagent kit according to claim 6, characterized in that, Further includes Lysis Buffer, DNA purification column, and solutions I / II / III.

8. A BRAF V600E mutation interpretation system, characterized in that, integrated: Raman spectrometer, with an excitation wavelength of 785±5nm and a power of 30±5mW; Data processing module, built-in 1188cm -1 and 1393cm -1 Bimodal peak identification algorithm; The output interface displays a "mutation positive" or "mutation negative" conclusion.

9. A method for prognostic assessment of thyroid cancer, characterized in that: The method described in claim 2 was used to detect BRAF V600E mutations in patient tissue samples; A positive mutation indicates the need to expand the surgical scope and initiate targeted therapy; a negative mutation indicates the need to use a standard surgical procedure.

10. An application of a BRAF V600E mutant probe, characterized in that, The probe described in claim 1 is used to prepare a recurrence monitoring reagent for thyroid cancer after surgery, and the treatment plan is adjusted by periodically detecting the mutation status in circulating tumor DNA.

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