Application of reagent for detecting methylated PTPRO gene in preparation of product for diagnosing cancer

Abstract

The invention discloses application of a reagent for detecting a methylated PTPRO gene in preparation of a product for diagnosing cancers. According to the present invention, the new marker such as the methylated PTPRO gene for cancer diagnosis is found, and by detecting the expression level of the marker, the non-invasive liquid diagnosis of cancer screening can be achieved, and different lung adenocarcinoma stages can be determined. The sample detected by the invention comprises saliva. And the saliva is easy to control and not easy to coagulate, so that the influence of anticoagulation treatment on the test result is eliminated. Meanwhile, saliva collection is non-invasive, a subject is basically free of pain and discomfort, and the method has repeatability and is safe and low in cost. According to the invention, DNA is extracted from the exosome to detect the methylated PTPRO gene, and a lipid membrane of the exosome can provide good protection for components carried by the exosome, so that the detection accuracy and stability can be maintained.

Description

Technical Field

[0001] This invention relates to the field of malignant tumors, and in particular to the application of reagents for detecting methylated PTPRO genes in the preparation of products for cancer diagnosis. Background Technology

[0002] Cancer has always threatened human health, with approximately 7 million people dying from it worldwide each year. Its onset is insidious, and its progression is rapid; by the time clinical symptoms appear or patients seek medical attention, it is often already in an advanced stage, with most patients dying within a few years of diagnosis. Lung cancer is the leading cause of death from malignant tumors in my country and worldwide, with lung adenocarcinoma (LUAD) being the most common pathological type, accounting for about 40% of all lung cancers. Despite significant advancements in surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy over the past few decades, the prognosis for lung cancer remains poor, with a 5-year survival rate of only 15%. Delayed diagnosis and distant metastasis are the main reasons for this poor survival rate. However, effective screening methods for early-stage LUAD are still lacking, and by the time it is detected, it is often already in an advanced stage. Currently, the diagnosis of LUAD typically relies on chest X-rays, radiologically or endoscopically guided biopsies for histological confirmation; these invasive techniques are unsuitable for screening asymptomatic individuals. Therefore, there is an urgent need to identify new, accurate, non-invasive biomarkers for the early diagnosis, monitoring, and prediction of disease progression and treatment response in LUAD patients. Monitoring cancer recurrence or treatment is a major challenge in clinical practice. Most cancer patients die from recurrence or metastasis, yet there are very few effective biomarkers for monitoring cancer recurrence. Detection relies mainly on imaging and laparoscopy, which have time and spatial limitations, making it difficult to detect recurrence in its early stages and to identify tumors hidden in recurrence or metastasis sites. Due to the prevalence of drug resistance in malignant tumors, the development of treatment plans and the monitoring of treatment efficacy are crucial; however, currently, there is a lack of effective biomarkers in clinical practice to guide treatment plan development and monitor treatment response.

[0003] Currently, the main diagnostic methods for lung adenocarcinoma include imaging examinations, cytopathology, and histopathology. Imaging examinations include X-ray examination, CT scan, MRI, PET, and ultrasound examination. However, these methods are generally inefficient and have low accuracy. Typical cytopathological examinations include cell biopsies, which require puncture or surgery to obtain tumor tissue samples from the patient, making them inconvenient and potentially harmful.

[0004] Liquid biopsy can overcome the shortcomings of traditional biopsies. Liquid biopsy refers to a detection method that uses simple bodily fluids to locate, study, and monitor the site of disease. Currently, blood is the most commonly used bodily fluid. However, blood has a complex composition, diverse origins, and fragmented information, which can cause nonspecificity and interference from hydrodynamics during the detection and analysis process. The large number of cells in blood with half-lives ranging from seconds to weeks or even months can also affect the analytical results. Currently, liquid biopsy mainly targets DNA fragments (ctDNA), circulating tumor cells (CTCs), and exosomes in plasma. CTCs and ctDNA have been studied earlier, but each has its limitations. For example, the content of CTCs and ctDNA is relatively low, and they cannot reflect the heterogeneity among tumor cells, thus significantly affecting the detection results.

[0005] Therefore, liquid biopsy methods for lung adenocarcinoma require further research and optimization. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide an application of a reagent for detecting methylated PTPRO genes in the preparation of cancer diagnostic products.

[0007] The objective of this invention is achieved through the following technical solution: Application of reagents for detecting methylated PTPRO genes in the preparation of cancer diagnostic products.

[0008] Furthermore, the reagent for detecting the methylated PTPRO gene is a reagent for detecting the methylated PTPRO gene in a sample; the sample includes at least one of body fluid and exosomes isolated from body fluid; further still, the body fluid includes at least one of blood or saliva; even further still, the body fluid is saliva; most preferably, the sample is exosomes isolated from saliva.

[0009] Furthermore, the reagent for detecting the methylated PTPRO gene is a reagent for detecting the amount of the methylated PTPRO gene.

[0010] Furthermore, the reagent for detecting the amount of methylated PTPRO gene includes at least one of DNA extraction reagent, bisulfite conversion reagent, methylation-specific polymerase chain reaction reagent, and agarose gel electrophoresis reagent. Furthermore, the reagents for the methylation-specific polymerase chain reaction include methylation-specific primer pairs; and even further, the methylation-specific primer pairs are... MSP-methylated-forward: 5'-CGTTTTTGGAGGATTTCGGGC-3'; and MSP-methylated-reverse: 5'-AAAACACGACTACGCTAACG-3'; Furthermore, the reagents for the methylation-specific polymerase chain reaction also include a non-methylation-specific primer pair; the non-methylation-specific primer pair is: MSP-unmethylated-forward: 5'-ATGTTTTTTGGAGGATTTTGGGT-3'; and MSP-unmethylated-reverse: 5'-ATACCCCATCACTACACAAACA-3'.

[0011] Furthermore, the amount of methylated PTPRO gene detected is a relative amount of methylated PTPRO gene detected; the relative amount of methylated PTPRO gene detected refers to whether the relative amount of methylated PTPRO gene is high or low. When the relative amount of methylated PTPRO gene is high, the diagnosis is positive, indicating that the subject has cancer; furthermore, the high relative amount of methylated PTPRO gene means that the target band is amplified by methylation-specific primer pairs; When the relative amount of methylated PTPRO gene is low, the diagnosis is negative; furthermore, the low relative amount of methylated PTPRO gene means that the methylation-specific primer pair did not amplify the target band, while the non-methylation-specific primer pair amplified the target band.

[0012] Furthermore, the cancers mentioned include, but are not limited to, lung cancer, esophageal cancer, stomach cancer, colorectal cancer, liver cancer, pancreatic cancer, ovarian cancer, cervical cancer, breast cancer, prostate cancer, testicular cancer, osteosarcoma, soft tissue sarcoma, and melanoma; even further, the cancer mentioned is lung cancer, which includes, but is not limited to, adenocarcinoma, squamous cell carcinoma, and small cell lung cancer; and still further, the cancer mentioned is lung adenocarcinoma.

[0013] Furthermore, the product includes a reagent kit.

[0014] The present invention has the following advantages and effects compared with the prior art: (1) This invention has discovered a new biomarker that can be used to diagnose cancer, especially lung adenocarcinoma: methylated PTPRO gene. By detecting the expression level of this biomarker, non-invasive liquid diagnosis of lung adenocarcinoma can be achieved.

[0015] (2) By detecting the level of expression of this marker, different stages of lung adenocarcinoma can also be determined.

[0016] (3) The sample tested in this invention includes saliva. Saliva is easy to handle, does not coagulate easily, and eliminates the influence of anticoagulation treatment on the test results. At the same time, saliva collection is non-invasive, with minimal pain and discomfort for the subject, and is repeatable, safe and inexpensive.

[0017] (4) The present invention detects the methylated PTPRO gene by extracting DNA from exosomes. The lipid membrane of exosomes can provide good protection for the components it carries, which is beneficial to maintaining the accuracy and stability of the detection.

[0018] (5) Exosomes actively take up effective components of tumor cells, which can better reflect the heterogeneity among tumor cells. Attached Figure Description

[0019] Figure 1 This is a comparison of PTPRO gene methylation levels in LUAD tissue and adjacent normal tissue.

[0020] Figure 2 This is a comparison chart of PTPRO gene methylation levels in LUAD at different cancer stages.

[0021] Figure 3 This is a gel electrophoresis result of lung adenocarcinoma cell lines and normal cell lines after treatment.

[0022] Figure 4 This is a gel electrophoresis result of processed salivary exosomes from some healthy volunteers and lung adenocarcinoma patients.

[0023] Figure 5 This is the ROC curve of the diagnostic efficacy of PTPRO gene methylation in the experimental group for lung adenocarcinoma.

[0024] Figure 6 This is the ROC curve of the diagnostic efficacy of PTPRO gene methylation in the validation group for lung adenocarcinoma. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0026] Example 1 Preliminary screening of clinical data in the database revealed that the methylation level of the PTPRO gene is associated with the stage of LUAD patients and can be used as a diagnostic biomarker; the specific experimental procedure is as follows: (1) Clinical data collection Transcriptomic and clinical information of LUAD samples and corresponding normal samples were obtained from the Cancer Genome Atlas (TCGA) database (https: / / portal.gdc.cancer.gov / ). Additionally, mean PTPRO gene methylation levels in LUAD tissues at various stages were collected from the LinkedOmics database (http: / / linkedomics.org / login.php#top).

[0027] (2) Analysis of PTPRO gene methylation levels in different stages of LUAD and adjacent normal tissues To assess the correlation between different LUAD stages and PTPRO gene methylation expression in adjacent normal tissues, we first analyzed TCGA data obtained from the UALCAN website (https: / / ualcan.path.uab.edu / ). Compared to adjacent normal tissues, the methylation level of the PTPRO gene in LUAD tissues was significantly higher (P < 0.001). Figure 1 (As shown). Furthermore, we found that the PTPRO gene methylation level in LUAD at different cancer stages was significantly increased compared to adjacent normal tissue (results are shown in Figure 1). Figure 2 (As shown).

[0028] Example 2 Cellular experiments revealed that the methylation level of the PTPRO gene in lung adenocarcinoma cell lines was higher than that in normal human bronchial epithelial cell lines, suggesting its potential as a diagnostic biomarker; the specific experimental procedure is as follows: (1) Sample preparation: Three lung adenocarcinoma cell lines were selected: human alveolar basal epithelial cells (A549 cells), human lung cancer cells (PC-9 cells), and human large cell lung cancer cells (NCI-H460 cells), as well as one immortalized bronchial epithelioid cell line (HBE cells) for culture. When the adherent cell density reached more than 80%, the cells were digested with trypsin, centrifuged to collect the precipitate, and prepared into a cell suspension for DNA extraction.

[0029] (2) DNA extraction: DNA was separated and extracted from cells using reagents from the EZ DNA Methylation-Direct Kit™ kit: the cell suspension was digested with protease for 30 minutes, and the reaction solution was centrifuged at 10,000 g / min for 5 minutes. The supernatant was the genomic product released after enzyme digestion, which could be used for gel electrophoresis to check the quality of the genome.

[0030] The protease digestion system includes 65 μL of M-digestion buffer, 5 μL of proteinase K solution, the sample, and ultrapure water to a final volume of 130 μL. The M-digestion buffer is prepared as follows: 20 mg of DNase A (SIGMA, catalog number R-4875) is dissolved in 2 mL of sodium acetate solution (0.01 mol sodium acetate, pH 5.0), boiled for 20 minutes, cooled to room temperature, and 1 mol Tris hydrochloric acid is added to adjust the pH to 7.5. The solution is then stored at -20°C. The proteinase K solution is prepared as follows: proteinase K is added to proteinase K storage buffer to a final concentration of 20 mg / mL, completely dissolved, and stored at -20°C. In a preferred embodiment, proteinase K is dissolved in sterile 50 mmol / L Tris (pH 8.0) and 1.5 mmol / L calcium acetate to prepare a 20 mg / mL proteinase K solution, which is then stored at -20°C.

[0031] (3) Bisulfite conversion: Use a pipette to aspirate 20 μL of the supernatant obtained in step (3) into a PCR tube, and then add 130 μL of CT conversion reagent solution. Mix the sample and centrifuge to ensure that there are no droplets on the tube cap and tube wall. Calculate the synergistic relationship between the maximum conversion capacity of the kit and the amount of DNA produced by the current digestion system. If there is an excess of DNA, incomplete conversion of sodium bisulfite may occur. Set the BIO-RAD S1000 temperature cycling thermostat (polymerase chain reaction instrument) to 98℃, place the PCR tube in it and time it for 8 minutes; set the water bath to 64℃, place the PCR tube in it and time it for 3.5 hours to obtain the conversion product; then place the PCR tube in a 4℃ refrigerator for later use (not exceeding 20 hours). The CT conversion reagent solution is prepared as follows: add 790 μL of M-dissolving buffer and 300 μL of M-dilution buffer to 1.78 g of sodium bisulfite powder, and then add 160 μL of M-reaction buffer and mix. Dissolve and shake at room temperature for 10 minutes or on a shaker for 10 minutes. It should be used immediately after preparation. The prepared CT conversion reagent is very sensitive to light, so minimize exposure to light. Storage conditions are as follows: overnight use at room temperature, within one week at 4°C, and within one month at -20°C. M-Dissolution buffer is 3 mol / L sodium hydroxide. M-Dilution buffer is autoclaved ultrapure water. M-Reaction buffer is 10 mmol / L hydroquinone.

[0032] Add 600 μL of the M-binding buffer provided in the bisulfite conversion kit to the inner tube of the collection tube (Zymo-Spin™ IC column). Transfer the conversion product to the inner tube containing the M-binding buffer. Cap and invert several times to mix. Centrifuge at ≥10000 g / min for 30 seconds. Aspirate the filtrate directly. Add 100 μL of M-wash buffer to the column. Centrifuge at 10000 g / min for 30 seconds. Aspirate the filtrate directly. Add 200 μL of M-desulfonate buffer to the column and incubate at room temperature (20–30 °C) for 15–20 minutes. After incubation, centrifuge at 10000 g / min for 30 seconds. Aspirate the filtrate directly. Add 200 μL of M-wash buffer to the column. Centrifuge at 10,000 g / min for 30 seconds, then add 200 μL of M-wash buffer to the column and centrifuge at 10,000 g / min for 30 seconds. Aspirate the filtrate directly. Place the column in a 1.5 mL centrifuge tube. Add 10 μL of M-elution buffer directly to the column matrix. Centrifuge at 10,000 g / min for 30 seconds to elute the DNA. Aspirate the filtrate directly. The DNA can be used immediately or stored at -20°C for later use. (1–4 μL of eluted DNA is recommended for each PCR test). M-desulfonate buffer: 10 mg / mL glycogen, 5 mmol ice-cold anhydrous ethanol. M-elution buffer: preheated ultrapure water.

[0033] (4) Methylation-specific polymerase chain reaction (MS-PCR): The DNA eluted in step (5) was subjected to a real-time fluorescent methylation-specific polymerase chain reaction (RT-PCR). The reaction system (total volume 25 μL) included the following components: 7.5 μL of ultrapure water, 12.5 μL of 2×QuantiFast SYBR Green PCR MasterMix premix, 2 μL of 10 μM primer pair, and 3 μL of bisulfite-converted DNA. The RT-PCR cycling parameters were: 1) 94℃ for 5 minutes; 2) 94℃ for 30 seconds; 3) 60℃ for 30 seconds; 4) return to step 2), repeat for 39 cycles; 5) 72℃ for 3 minutes; 6) maintain the temperature at 12℃.

[0034] In fluorescent methylation-specific polymerase chain reaction (PCR), we used self-designed and validated methylation-specific primer pairs or non-methylation-specific primer pairs, achieving high detection sensitivity and specificity. M primers (MSP-methylated-forward and MSP-methylated-reverse) are methylated-specific primers, meaning they specifically amplify PCR products when CpG islands in the gene are methylated. U primers (MSP-unmethylated-forward and MSP-unmethylated-reverse) are unmethylated-specific primers, specifically amplifying unmethylated bands when CpG islands in the gene are at low methylation levels. This set of M and U primer pairs recognizes a total of 10 methylation sites in the CpG region of this gene, and the same primers can be used for different samples (whether for lung adenocarcinoma, esophageal tumor, or other tumors).

[0035] The sequence of MSP-methylated-forward is: 5'-CGTTTTTGGAGGATTTCGGGC-3'; The sequence of MSP-methylated-reverse is: 5'-AAAACACGACTACGCTAACG-3'; The sequence of MSP-unmethylated-forward is: 5'-ATGTTTTTTGGAGGATTTTGGGT-3'; The sequence of MSP-unmethylated-reverse is: 5'-ATACCCCATCACTACACAAACA-3'.

[0036] (5) Agarose gel electrophoresis: To prepare 1% agarose gel: Weigh 0.5 g of agarose using an electronic balance and place it in an Erlenmeyer flask. Add 50 mL of 1×tris-acetic acid (1×TAE), seal the flask opening with kraft paper, and microwave on medium heat until boiling for about 3 minutes until the agarose is completely dissolved. Shake well to obtain a 1.0% agarose gel solution. Then add 0.5 μg / mL of ethidium bromide and shake well.

[0037] Gel preparation: Seal the glass plate and the edges of the inner tank to form a mold. Place the inner tank horizontally and position the comb in a fixed position. Carefully pour the agarose gel solution, cooled to approximately 65°C, into the glass plate of the inner tank, allowing the gel to spread slowly until a uniform gel layer forms on the entire surface of the glass plate. Let it stand at room temperature until the gel is completely solidified. Gently pull the comb vertically to remove the gel tape. Place the gel and inner tank into the electrophoresis tank, and add 1×tris acetic acid electrophoresis buffer until it covers the gel plate by 1-2 mm.

[0038] Sample loading: Mix the DNA sample and loading buffer on the sample plate. Using a 10 μL micropipette, add the sample to the sample wells of the gel plate. Replace the pipette tip after each sample to prevent contamination. Do not damage the gel surface around the sample wells during loading. (Note: Remember the loading order before loading the samples).

[0039] Electrophoresis: Immediately after sample loading, electrophoresis is performed on the gel plate at 70 volts for 30 minutes, with the sample moving from the negative electrode (black) to the positive electrode (red). Electrophoresis is stopped when the bromophenol blue in the loading buffer reaches approximately 1 cm from the bottom edge of the gel plate.

[0040] Electrophoresis results of methylated and unmethylated PTPRO gene sequences in A549, PC-9, NCI-H460, and HBE cells are as follows: Figure 3 As shown, ddH2O was used as a negative control. The results showed that significant PTPRO gene methylation was detected in A549 cells, PC-9 cells, and NCI-H460 cells, while no PTPRO gene methylation was detected in HBE cells.

[0041] Example 3 The methylation status of the PTPRO gene in the saliva and salivary exosomes of patients with lung adenocarcinoma was detected. Saliva samples were collected from 27 patients with early-stage lung adenocarcinoma and 19 healthy volunteers in Guangzhou, Guangdong Province, with strict control over age, sex, and smoking status among patients and healthy volunteers. The methylation status of the PTPRO gene in different samples was effectively monitored using methylation-specific polymerase chain reaction (MS-PCR), and the diagnostic ability of PTPRO gene methylation for lung adenocarcinoma was assessed using ROC curve analysis. Specific experiments are as follows: (1) Collection, preservation and transportation of saliva 1) Collection: Between 8 and 10 a.m., the non-irritating droplet method was used to collect whole saliva from subjects who met the following conditions in a quiet and comfortable environment: 1) fasting and no strenuous exercise; 2) instructing subjects to rinse their mouths with about 20 to 60 mL of mouthwash to remove food residue and other debris, spit out any remaining liquid, lower their heads slightly, sit quietly and wait for saliva to flow out naturally, collect their non-irritating whole saliva in a collection dish, preferably enough to cover the entire culture dish, about 250 to 900 µL, and then quickly aliquot it into sterile storage tubes.

[0042] 2) Preservation: After centrifugation in a preservation tube, the collected saliva can be stored for a long time at -80℃.

[0043] 3) Transportation: If transportation is required, it can be placed in a transport box to ensure that subsequent experiments are not affected by various microorganisms or enzymes in saliva.

[0044] Over the years, the inventor has collected a large number of saliva samples from cancer patients and healthy individuals for exosome testing from multiple hospitals in China (all samples were collected, preserved, and transported according to the above standards). All patients were followed up every three months, and their survival status and recurrence were recorded in detail, establishing complete case follow-up data.

[0045] (2) Extraction, preservation and transportation of salivary exosomes 1) Extraction of salivary exosomes: Take an equal volume of 500 µL of saliva obtained in step 1 (saliva without centrifugation), centrifuge at 300 g for 10 min, transfer the supernatant to a new centrifuge tube, centrifuge at 2000 g for 10 min, transfer the supernatant to a new centrifuge tube, centrifuge at 5000 g for 20 min, and transfer the supernatant obtained by centrifugation to a new 1.5 mL EP tube to obtain approximately 450–470 µL of clear supernatant; add exosome precipitation reagent at a volume ratio of clear supernatant to exosome precipitation reagent = 250:63, mix well, incubate at 4°C overnight, centrifuge at 1500 g at 4°C for 30 min, centrifuge at 3000 g for 5 min, discard the supernatant, collect the precipitate, and resuspend the precipitate in an appropriate amount of 1× exosome suspension (1×PBS in this example) to obtain exosome resuspension.

[0046] Based on Nanosight analysis of the corresponding number of exosomes, the concentration obtained from 500 µL of saliva is 1–10 × 10⁻⁶. 6 Particles / mL of exocrine body suspension.

[0047] 2) Preservation and transportation of salivary exosomes The obtained exosomes can be stored for a long time in a -80°C freezer in storage tubes, and the DNA, RNA, proteins, etc. contained therein can be preserved relatively stably.

[0048] (3) DNA extraction: DNA was separated and extracted from cells using reagents from the EZ DNA Methylation-Direct Kit™ kit: the exosomes obtained in step (2) were digested with protease for 30 minutes, and the reaction solution was centrifuged at 10000 g / min for 5 minutes. The supernatant was the genomic product released after enzyme digestion, which can be used for gel electrophoresis to check the quality of the genome.

[0049] The protease digestion system includes 65 μL of M-digestion buffer, 5 μL of proteinase K solution, the sample, and ultrapure water to a final volume of 130 μL. The M-digestion buffer is prepared as follows: 20 mg of DNase A (SIGMA, catalog number R-4875) is dissolved in 2 mL of sodium acetate solution (0.01 mol sodium acetate, pH 5.0), boiled for 20 minutes, cooled to room temperature, and 1 mol Tris hydrochloric acid is added to adjust the pH to 7.5. The solution is then stored at -20°C. The proteinase K solution is prepared as follows: proteinase K is added to proteinase K storage buffer to a final concentration of 20 mg / mL, completely dissolved, and stored at -20°C. In a preferred embodiment, proteinase K is dissolved in sterile 50 mmol / L Tris (pH 8.0) and 1.5 mmol / L calcium acetate to prepare a 20 mg / mL proteinase K solution, which is then stored at -20°C.

[0050] (3) Bisulfite conversion: Use a pipette to aspirate 20 μL of the supernatant obtained in step (3) into a PCR tube, and then add 130 μL of CT conversion reagent solution. Mix the sample and centrifuge to ensure that there are no droplets on the tube cap and tube wall. Calculate the synergistic relationship between the maximum conversion capacity of the kit and the amount of DNA produced by the current digestion system. If there is an excess of DNA, incomplete conversion of sodium bisulfite may occur. Set the BIO-RAD S1000 temperature cycling thermostat (polymerase chain reaction instrument) to 98℃, place the PCR tube in it and time it for 8 minutes; set the water bath to 64℃, place the PCR tube in it and time it for 3.5 hours to obtain the conversion product; then place the PCR tube in a 4℃ refrigerator for later use (not exceeding 20 hours). The CT conversion reagent solution is prepared as follows: add 790 μL of M-dissolving buffer and 300 μL of M-dilution buffer to 1.78 g of sodium bisulfite powder, and then add 160 μL of M-reaction buffer and mix. Dissolve and shake at room temperature for 10 minutes or on a shaker for 10 minutes. It should be used immediately after preparation. The prepared CT conversion reagent is very sensitive to light, so minimize exposure to light. Storage conditions are as follows: overnight use at room temperature, within one week at 4°C, and within one month at -20°C. M-Dissolution buffer is 3 mol / L sodium hydroxide. M-Dilution buffer is autoclaved ultrapure water. M-Reaction buffer is 10 mmol / L hydroquinone.

[0051] Add 600 μL of the M-binding buffer provided in the bisulfite conversion kit to the inner tube of the collection tube (Zymo-Spin™ IC column). Transfer the conversion product to the inner tube containing the M-binding buffer. Cap and invert several times to mix. Centrifuge at ≥10000 g / min for 30 seconds. Aspirate the filtrate directly. Add 100 μL of M-wash buffer to the column. Centrifuge at 10000 g / min for 30 seconds. Aspirate the filtrate directly. Add 200 μL of M-desulfonate buffer to the column and incubate at room temperature (20–30 °C) for 15–20 minutes. After incubation, centrifuge at 10000 g / min for 30 seconds. Aspirate the filtrate directly. Add 200 μL of M-wash buffer to the column. Centrifuge at 10,000 g / min for 30 seconds, then add 200 μL of M-wash buffer to the column and centrifuge at 10,000 g / min for 30 seconds. Aspirate the filtrate directly. Place the column in a 1.5 mL centrifuge tube. Add 10 μL of M-elution buffer directly to the column matrix. Centrifuge at 10,000 g / min for 30 seconds to elute the DNA. Aspirate the filtrate directly. The DNA can be used immediately or stored at -20°C for later use. (1–4 μL of eluted DNA is recommended for each PCR test). M-desulfonate buffer: 10 mg / mL glycogen, 5 mmol ice-cold anhydrous ethanol. M-elution buffer: preheated ultrapure water.

[0052] (4) Methylation-specific polymerase chain reaction (MS-PCR): The DNA eluted in step (5) was subjected to a real-time fluorescent methylation-specific polymerase chain reaction (RT-PCR). The reaction system (total volume 25 μL) included the following components: 7.5 μL of ultrapure water, 12.5 μL of 2×QuantiFast SYBR Green PCR MasterMix premix, 2 μL of 10 μM primer pair, and 3 μL of bisulfite-converted DNA. The RT-PCR cycling parameters were: 1) 94℃ for 5 minutes; 2) 94℃ for 30 seconds; 3) 60℃ for 30 seconds; 4) return to step 2), repeat for 39 cycles; 5) 72℃ for 3 minutes; 6) maintain the temperature at 12℃.

[0053] In fluorescent methylation-specific polymerase chain reaction (PCR), we used self-designed and validated methylation-specific primer pairs or non-methylation-specific primer pairs, achieving high detection sensitivity and specificity. M primers (MSP-methylated-forward and MSP-methylated-reverse) are methylated-specific primers, meaning they specifically amplify PCR products when CpG islands in the gene are methylated. U primers (MSP-unmethylated-forward and MSP-unmethylated-reverse) are unmethylated-specific primers, specifically amplifying unmethylated bands when CpG islands in the gene are at low methylation levels. This set of M and U primer pairs recognizes a total of 10 methylation sites in the CpG region of this gene, and the same primers can be used for different samples (whether for lung adenocarcinoma, esophageal tumor, or other tumors).

[0054] The sequence of MSP-methylated-forward is: 5'-CGTTTTTGGAGGATTTCGGGC-3'; The sequence of MSP-methylated-reverse is: 5'-AAAACACGACTACGCTAACG-3'; The sequence of MSP-unmethylated-forward is: 5'-ATGTTTTTTGGAGGATTTTGGGT-3'; The sequence of MSP-unmethylated-reverse is: 5'-ATACCCCATCACTACACAAACA-3'.

[0055] (5) Agarose gel electrophoresis: To prepare 1% agarose gel: Weigh 0.5 g of agarose using an electronic balance and place it in an Erlenmeyer flask. Add 50 mL of 1×tris-acetic acid (1×TAE), seal the flask opening with kraft paper, and microwave on medium heat until boiling for about 3 minutes until the agarose is completely dissolved. Shake well to obtain a 1.0% agarose gel solution. Then add 0.5 μg / mL of ethidium bromide and shake well.

[0056] Gel preparation: Seal the glass plate and the edges of the inner tank to form a mold. Place the inner tank horizontally and position the comb in a fixed position. Carefully pour the agarose gel solution, cooled to approximately 65°C, into the glass plate of the inner tank, allowing the gel to spread slowly until a uniform gel layer forms on the entire surface of the glass plate. Let it stand at room temperature until the gel is completely solidified. Gently pull the comb vertically to remove the gel tape. Place the gel and inner tank into the electrophoresis tank, and add 1×tris acetic acid electrophoresis buffer until it covers the gel plate by 1-2 mm.

[0057] Sample loading: Mix the DNA sample and loading buffer on the sample plate. Using a 10 μL micropipette, add the sample to the sample wells of the gel plate. Replace the pipette tip after each sample to prevent contamination. Do not damage the gel surface around the sample wells during loading. (Note: Remember the loading order before loading the samples).

[0058] Electrophoresis: Immediately after sample loading, electrophoresis is performed on the gel plate at 70 volts for 30 minutes, with the sample moving from the negative electrode (black) to the positive electrode (red). Electrophoresis is stopped when the bromophenol blue in the loading buffer reaches approximately 1 cm from the bottom edge of the gel plate.

[0059] Electrophoresis results of PTPRO gene methylation in salivary exosomes from some healthy individuals and lung adenocarcinoma patients in the Guangzhou sample data group are as follows: Figure 4 As shown in Table 1, the statistical results of the Guangzhou sample data group demonstrate that methylation-specific polymerase chain reaction (MS-PCR) can effectively monitor the methylation status of PTPRO in different samples. The methylation detection rate of the PTPRO gene in salivary exosomes of healthy individuals was significantly lower than that of lung adenocarcinoma patients. Furthermore, the ROC curve analysis results are shown below. Figure 5 As shown, when using salivary exosome PTPRO gene methylation as a marker to distinguish between lung adenocarcinoma patients and healthy individuals, the AUC value was 0.812, the sensitivity was 74.1%, and the specificity was 96.7%.

[0060] Table 1

[0061] Example 4 Saliva samples from 31 patients with early-stage lung adenocarcinoma and 21 healthy volunteers in Shantou City, Guangdong Province, were collected as a validation set for further validation. Age, sex, and smoking status were strictly controlled between patients and healthy volunteers. Methylation-specific polymerase chain reaction (MS-PCR) was used to effectively monitor the methylation status of the PTPRO gene in different samples, and ROC curve analysis was used to verify the diagnostic ability of detecting PTPRO gene methylation for lung adenocarcinoma.

[0062] The experimental method was the same as in Example 3, and the detection results are shown in Table 2. The ROC curve analysis results are as follows: Figure 6 As shown, when using PTPRO gene methylation in salivary exosomes as a biomarker to distinguish between lung adenocarcinoma patients and healthy individuals, the AUC value was 0.869, the sensitivity was 67.7%, and the specificity was 90.5%. Therefore, PTPRO gene methylation in salivary exosomes can serve as an important candidate molecular biomarker for the early diagnosis of lung adenocarcinoma.

[0063] Table 2

[0064] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of reagents for detecting methylated PTPRO genes in the preparation of cancer diagnostic products.

2. The application according to claim 1, characterized in that, The cancers mentioned include lung cancer.

3. The application according to claim 1, characterized in that, The reagent for detecting methylated PTPRO genes is a reagent for detecting methylated PTPRO genes in a sample; the sample includes at least one of body fluids and exosomes isolated from body fluids.

4. The application according to claim 3, characterized in that, The bodily fluids include at least one of blood or saliva.

5. The application according to claim 3, characterized in that, The sample was an exosome isolated from saliva.

6. The application according to claim 1, characterized in that, The reagent for detecting methylated PTPRO genes is a reagent for detecting the amount of methylated PTPRO genes.

7. The application according to claim 6, characterized in that, The reagent for detecting the amount of methylated PTPRO gene includes at least one of DNA extraction reagent, bisulfite conversion reagent, methylation-specific polymerase chain reaction reagent, and agarose gel electrophoresis reagent.

8. The application according to claim 7, characterized in that, The reagents for the methylation-specific polymerase chain reaction include methylation-specific primer pairs; the methylation-specific primer pairs are... MSP-methylated-forward: 5'-CGTTTTTGGAGGATTTCGGGC-3'; and MSP-methylated-reverse: 5'-AAAACACGACTACGCTAACG-3'.

9. The application according to claim 8, characterized in that, The reagents for the methylation-specific polymerase chain reaction also include a non-methylation-specific primer pair; the non-methylation-specific primer pair is: MSP-unmethylated-forward: 5'-ATGTTTTTTGGAGGATTTTGGGT-3'; and MSP-unmethylated-reverse: 5'-ATACCCCATCACTACACAAACA-3'.

10. The application according to claim 1, characterized in that, The products mentioned include reagent kits.

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