Method and kit for measuring methylation levels of bladder cancer biomarkers

Abstract

Provided are a method and kit for measuring the methylation levels of bladder cancer biomarkers. The bladder cancer biomarkers include a Twist1 gene and an SPN gene. By means of measuring the methylation levels of the Twist1 gene and the SPN gene, the present application can accurately detect bladder cancer.

Description

A method and kit for detecting methylation levels of bladder cancer biomarkers Technical Field

[0001] The present application relates to the field of biology. Specifically, the present application relates to a method and a kit for detecting the methylation level of a bladder cancer biomarker. Background Art

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system, ranking sixth among men and 17th among women. Bladder cancer varies by region, race, and gender, and can occur in all age groups, with a peak incidence between 50 and 70 years old. The incidence rate in men is significantly higher than in women, approximately three to four times higher. For bladder cancer of the same stage, women have higher recurrence and mortality rates than men.

[0003] Based on histopathology, over 90% of bladder cancer cases present with urothelial (transitional cell) carcinoma, 5% with squamous cell carcinoma, and less than 2% with adenocarcinoma. Based on the depth of invasion into the bladder wall, bladder cancer can be categorized as non-muscle-invasive bladder cancer (NMIBC) or muscle-invasive bladder cancer (MIBC). Among patients newly diagnosed with bladder cancer, 70% to 85% present with NMIBC, and 15% to 30% with MIBC. NMIBC is classified into the following stages: Ta (papillary), T1 (lamina propria invasion), and carcinoma in situ. Ta accounts for 70% of patients, T1 accounts for approximately 20%, and carcinoma in situ accounts for approximately 10%. MIBC is classified into the following stages: T2, T3, and T4. Up to 80% of NMIBC patients relapse within 5 years; 30% of Ta patients progress to MIBC; while T1 and carcinoma in situ are more likely to progress to MIBC. Transurethral resection of the bladder tumor is considered the standard treatment for NMIBC, with intravesical therapy determined by the risk of recurrence. Neoadjuvant chemotherapy combined with radical cystectomy is the standard treatment for patients with MIBC.

[0004] The development and progression of bladder cancer is a complex, multifactorial process, with both intrinsic genetic factors and extrinsic environmental factors having significant influence. Smoking and long-term exposure to industrial chemicals are currently the two most established external risk factors for bladder cancer. Approximately 50% of bladder cancer patients have a history of smoking. Smokers have a two- to three-fold increased risk of bladder cancer, with the risk proportional to the intensity and duration of smoking. Long-term occupational exposure to industrial chemicals is another significant risk factor, including aromatic amines, polycyclic aromatic hydrocarbons, chlorinated hydrocarbons, β-naphthylamine, and 4-aminobiphenyl. Furthermore, the development of bladder cancer is also linked to heredity and genetic abnormalities. The malignant transformation of normal bladder cells begins with alterations in the cell's DNA. Those with a family history of bladder cancer have a two-fold increased risk of bladder cancer. Therefore, accurate diagnosis and assessment of early-stage bladder cancer patients are crucial in clinical practice to increase the chances of bladder preservation surgery and improve overall survival.

[0005] Currently, bladder cancer is diagnosed primarily through cystoscopy and biopsy, as well as urine cytology. Cystoscopy and biopsy are the most reliable methods for diagnosing bladder cancer, but they are invasive, expensive, and can cause pain, bleeding, urinary tract infections, and other complications. Furthermore, they can sometimes be difficult to detect tumors in secluded areas of the bladder. Cytology is a noninvasive test that directly identifies shed tumor cells in urine. It is simple to use, inexpensive, and has high specificity (85%-100%), but low sensitivity (13%-75%) and diagnostic efficiency, particularly for low-grade bladder cancer (16%). Recently, several noninvasive methods, such as NMP-22, bladder tumor antigen, and FISH, have been shown to improve the sensitivity of urine cytology. However, due to limited specificity or sensitivity, the markers proposed to date have not been widely adopted in routine clinical practice. Therefore, there is a need for novel, noninvasive, low-cost, and highly accurate diagnostic and monitoring methods to guide clinical practice. Summary of the Invention

[0006] The main purpose of this application is to provide a simple, rapid, low-cost, highly accurate and non-invasive method for bladder cancer detection.

[0007] In order to achieve the above objectives, the present disclosure is implemented through the following technical solutions:

[0008] The inventors of this disclosure have discovered that the SPN gene is significantly associated with bladder cancer. The degree of methylation at its methylation sites is strongly correlated with the development of bladder cancer. However, no research has yet been found on the SPN gene for bladder cancer detection. Therefore, this disclosure is the first to use the SPN gene as a biomarker for bladder cancer detection. Furthermore, the inventors discovered that combining the SPN gene with the Twist1 gene as a biomarker for bladder cancer detection can significantly improve the accuracy, sensitivity, and specificity of detection, which is of great significance for the early diagnosis of bladder cancer.

[0009] In one aspect of the present disclosure, a biomarker is provided. According to an embodiment of the present disclosure, the biomarker includes Twist1 gene and SPN gene.

[0010] The inventors of the present disclosure have discovered that the methylation levels of the Twist1 gene and the SPN gene are significantly correlated with the occurrence of bladder cancer. By detecting the methylation levels of these two genes, bladder cancer can be accurately diagnosed with high accuracy, high sensitivity, and high specificity. In addition, the detection method is simple, rapid, low-cost, and easy to sample, which can achieve non-invasive early diagnosis of bladder cancer and has good application prospects.

[0011] According to an embodiment of the present disclosure, the biomarkers include methylation sites in the Twist1 gene target region and methylation sites in the SPN gene target region; taking GRCh38.p14 as the reference genome, the Twist1 gene target region is selected from the Chr7:19118311-19118422 negative chain, and the SPN gene target region is selected from the Chr16:29664517-29664618 positive chain.

[0012] According to an embodiment of the present disclosure, the methylation site of the Twist1 gene target region is selected from at least one of the following: Chr7:19118316, Chr7:19118321, Chr7:19118379, Chr7:19118403, Chr7:19118408 and Chr7:19118419; the methylation site of the SPN gene target region is selected from at least one of the following: Chr16:29664525, Chr16:29664538, Chr16:29664575, Chr16:29664578, Chr16:29664607 and Chr16:29664614.

[0013] In yet another aspect, the present disclosure provides a primer set. According to an embodiment of the present disclosure, the primer set is used to detect the aforementioned biomarkers. The primer set comprises: a first primer set, wherein the nucleotide sequences of the primers in the first primer set are shown in SEQ ID NOs: 1 and 2, respectively; and a second primer set, wherein the nucleotide sequences of the primers in the second primer set are shown in SEQ ID NOs: 4 and 5, respectively. Thus, the primer set of the present disclosure can be used to specifically amplify methylation sites of the Twist1 and SPN genes, thereby diagnosing bladder cancer.

[0014] In yet another aspect, the present disclosure provides a probe set. According to an embodiment of the present disclosure, the probe set is used to detect the aforementioned biomarkers. The probe set includes a first probe having a nucleotide sequence as shown in SEQ ID NO: 3; and a second probe having a nucleotide sequence as shown in SEQ ID NO: 6. Thus, the probe set disclosed herein can be used to detect the Twist1 gene and the SPN gene by fluorescent quantitative PCR, thereby diagnosing bladder cancer.

[0015] According to an embodiment of the present disclosure, the 5' end of the first probe is labeled with a FAM fluorescent reporter group, and the 3' end is labeled with an MGB fluorescent quencher group; the 5' end of the second probe is labeled with a ROX fluorescent reporter group, and the 3' end is labeled with an MGB fluorescent quencher group.

[0016] In yet another aspect, the present disclosure provides a kit. According to embodiments of the present disclosure, the kit is used to detect the aforementioned biomarkers, comprising at least one of the following: the aforementioned primer set and the aforementioned probe set. Thus, the kit can be used to detect methylation levels of the Twist1 and SPN genes, thereby facilitating the diagnosis of bladder cancer.

[0017] According to an embodiment of the present disclosure, the kit further includes at least one of the following: a sample collection container, a DNA extraction reagent, a methylation conversion reagent, a fluorescent quantitative PCR detection reagent, a primer for detecting the β-actin gene, and a probe for detecting the β-actin gene.

[0018] According to an embodiment of the present disclosure, the sample collection container is used to collect samples, and the capacity of the sample collection container is 1 to 10 mL.

[0019] According to an embodiment of the present disclosure, the 5' end of the probe for detecting the β-actin gene is labeled with a VIC fluorescent reporter group, and the 3' end is labeled with an MGB fluorescent quencher group.

[0020] In another aspect of the present disclosure, the present disclosure provides the use of a reagent for detecting the aforementioned biomarkers in the preparation of a detection product. According to an embodiment of the present disclosure, the detection product is used for diagnosing bladder cancer.

[0021] According to an embodiment of the present disclosure, the reagent for detecting a biomarker includes the aforementioned primer set and / or the aforementioned probe set.

[0022] According to an embodiment of the present disclosure, the detection product includes a kit, a reagent strip or a chip.

[0023] According to an embodiment of the present disclosure, the kit is selected from the kit described above.

[0024] In another aspect of the present disclosure, a diagnostic method for detecting methylation levels of bladder cancer biomarkers is provided. According to an embodiment of the present disclosure, the diagnostic method comprises detecting the aforementioned biomarkers in a biological sample using the aforementioned primer set and probe set. Thus, the disclosed method can accurately detect methylation levels of the Twist1 and SPN genes in bladder cancer.

[0025] In another aspect of the present disclosure, a device for diagnosing bladder cancer is provided. According to an embodiment of the present disclosure, the device for diagnosing bladder cancer includes: a detection unit adapted to detect the methylation level of the aforementioned biomarkers and obtain a detection result; and an analysis unit adapted to diagnose bladder cancer based on the detection result. As such, the device of the present disclosure offers simple and rapid operation, high detection accuracy, low cost, and convenient sampling, enabling non-invasive early diagnosis of bladder cancer and promising application prospects.

[0026] According to an embodiment of the present disclosure, the detection unit contains a detection device; the detection device includes a fluorescence quantitative PCR instrument.

[0027] According to an embodiment of the present disclosure, the detection unit further contains a DNA extraction device.

[0028] According to an embodiment of the present disclosure, the analysis unit is suitable for diagnosing bladder cancer based on the following judgment method: under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated positive; if Ct(Twist1)-Ct(β-actin)>9.4 or there is no amplification curve in the Twist1 gene target region, it is judged that the Twist1 gene is methylated negative; under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated negative. Under the premise that the amplification curve is S-shaped and Ct(β-actin)≤35, if the amplification curve of the target region of the SPN gene is S-shaped and Ct(SPN)-Ct(β-actin)≤10.1, the SPN gene is judged to be positive for methylation; if Ct(SPN)-Ct(β-actin)>10.1 or there is no amplification curve in the target region of the SPN gene, the SPN gene is judged to be negative for methylation; when at least one of the Twist1 gene and the SPN gene is positive for methylation, the patient is judged to have bladder cancer; when both the Twist1 gene and the SPN gene are negative for methylation, the patient is judged to not have bladder cancer.

[0029] In yet another aspect of the present disclosure, an analysis system for diagnosing bladder cancer is provided. According to an embodiment of the present disclosure, the analysis system for diagnosing bladder cancer includes: a data acquisition module configured to acquire detection results of the aforementioned biomarker methylation levels in a biological sample; and an analysis module configured to diagnose bladder cancer based on the detection results. Thus, the analysis system of the present disclosure can accurately diagnose bladder cancer.

[0030] According to an embodiment of the present disclosure, the analysis module is configured to diagnose bladder cancer based on the following judgment method: under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated positive; if Ct(Twist1)-Ct(β-actin)>9.4 or there is no amplification curve in the Twist1 gene target region, it is judged that the Twist1 gene is methylated negative; under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated negative. Under the premise that the amplification curve is S-shaped and Ct(β-actin)≤35, if the amplification curve of the target region of the SPN gene is S-shaped and Ct(SPN)-Ct(β-actin)≤10.1, the SPN gene is judged to be positive for methylation; if Ct(SPN)-Ct(β-actin)>10.1 or there is no amplification curve in the target region of the SPN gene, the SPN gene is judged to be negative for methylation; when at least one of the Twist1 gene and the SPN gene is positive for methylation, the patient is judged to have bladder cancer; when both the Twist1 gene and the SPN gene are negative for methylation, the patient is judged to not have bladder cancer.

[0031] In another aspect of the present disclosure, an electronic device is provided. According to an embodiment of the present disclosure, the electronic device includes a memory and a processor. The memory stores a program executable by the processor. When executed by the processor, the program diagnoses bladder cancer based on the methylation levels of the aforementioned biomarkers in a biological sample. Thus, the electronic device of the present disclosure can accurately diagnose bladder cancer.

[0032] According to an embodiment of the present disclosure, when the program is executed by the processor, bladder cancer is diagnosed based on the following judgment method: under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated positive; if Ct(Twist1)-Ct(β-actin)>9.4 or there is no amplification curve in the Twist1 gene target region, it is judged that the Twist1 gene is methylated negative; under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated negative. Under the premise that the gene amplification curve is S-shaped and Ct(β-actin)≤35, if the amplification curve of the target region of the SPN gene is S-shaped and Ct(SPN)-Ct(β-actin)≤10.1, the SPN gene is judged to be positive for methylation. If Ct(SPN)-Ct(β-actin)>10.1 or there is no amplification curve in the target region of the SPN gene, the SPN gene is judged to be negative for methylation. When at least one of the Twist1 gene and the SPN gene is positive for methylation, the patient is judged to have bladder cancer. When both the Twist1 gene and the SPN gene are negative for methylation, the patient is judged to not have bladder cancer.

[0033] In another aspect, the present disclosure provides a computer-readable storage medium. According to an embodiment of the present disclosure, the computer-readable storage medium stores one or more programs, which can be executed by one or more processors to enable diagnosis of bladder cancer based on the methylation levels of the aforementioned biomarkers in a biological sample. Thus, the computer-readable storage medium of the present disclosure can accurately diagnose bladder cancer.

[0034] According to an embodiment of the present disclosure, when the one or more programs are executed by one or more processors, the diagnosis of bladder cancer is based on the following judgment method: under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated positive; if Ct(Twist1)-Ct(β-actin)>9.4 or there is no amplification curve in the Twist1 gene target region, it is judged that the Twist1 gene is methylated negative; under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated negative. Under the premise that the amplification curve of the β-actin gene is S-shaped and Ct(β-actin)≤35, if the amplification curve of the target region of the SPN gene is S-shaped and Ct(SPN)-Ct(β-actin)≤10.1, the SPN gene is judged to be methylation-positive; if Ct(SPN)-Ct(β-actin)>10.1 or there is no amplification curve of the target region of the SPN gene, the SPN gene is judged to be methylation-negative; when at least one of the Twist1 gene and the SPN gene is methylation-positive, the patient is judged to have bladder cancer; when both the Twist1 gene and the SPN gene are methylation-negative, the patient is judged to not have bladder cancer.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] (1) Methylation abnormalities are an early event in the development of tumors. This disclosure can detect the methylation levels of the Twist1 and SPN genes to diagnose early bladder cancer, effectively reducing the incidence and mortality of bladder cancer. The method is easy to use. After obtaining a urine sample collection tube, the user can complete the urine sample collection at home and then send it by express delivery to be tested by professionals.

[0037] (2) The disclosed method for detecting the methylation levels of the Twist1 gene and the SPN gene is completely non-invasive, uses urine as the test sample, is simple to sample and easy to obtain, does not cause any pain or impact to the patient, and is highly acceptable to patients.

[0038] (3) The present disclosure uses 1 to 10 mL of whole urine samples for testing, especially 2 mL of whole urine samples for testing, and can still achieve high levels of sensitivity and specificity, which is applicable to small volume sample testing.

[0039] (4) The present invention designs specific primers and probes, and adopts a method of jointly detecting the methylation of the Twist1 gene and the SPN gene, which greatly improves the overall detection accuracy, sensitivity and specificity. The detection sensitivity is 92.1% and the specificity is 94.6%, avoiding the problems of low sensitivity and specificity existing in single gene methylation detection.

[0040] (5) Among the bladder cancer target genes used in the present disclosure, the Twist1 gene probe is labeled with FAM, the SPN gene probe is labeled with ROX, and the control β-actin gene probe is labeled with VIC, which can realize multiple single-tube detection. Compared with single-plex detection, it reduces reagent consumption, reduces consumables costs, reduces the number of operating steps for experimenters, and reduces the experimental error rate.

[0041] Additional aspects and advantages of the present disclosure will be set forth in part in the following description and, in part, will be obvious from the following description, or may be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0043] Figure 1 shows the ROC curve of the Twist1 gene detection results;

[0044] FIG2 shows the ROC curve of the SPN gene detection results. DETAILED DESCRIPTION

[0045] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0046] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of the features. Furthermore, in the description of this disclosure, unless otherwise specified, "plurality" means two or more.

[0047] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0048] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present disclosure, but not excluding other contents.

[0049] The present disclosure proposes biomarkers, primer sets, probe sets, kits and their applications, diagnostic methods for detecting methylation levels of bladder cancer biomarkers, devices and analysis systems for diagnosing bladder cancer, electronic devices and computer-readable storage media, each of which will be described in detail below.

[0050] Biomarkers

[0051] In one aspect, the present disclosure provides a biomarker. According to an embodiment of the present disclosure, the biomarker includes the Twist1 gene and the SPN gene. The inventors of the present disclosure have discovered that the methylation levels of the Twist1 and SPN genes are significantly correlated with the occurrence of bladder cancer. By detecting the methylation levels of these two genes, bladder cancer can be accurately diagnosed with high accuracy, sensitivity, and specificity. Furthermore, the detection method is simple, rapid, low-cost, and convenient for sampling, enabling non-invasive early diagnosis of bladder cancer and promising application prospects.

[0052] In another aspect of the present disclosure, another biomarker is proposed. According to an embodiment of the present disclosure, the biomarker includes methylation sites in the target region of the Twist1 gene and methylation sites in the target region of the SPN gene; using GRCh38.p14 as the reference genome, the target region of the Twist1 gene is selected from the negative chain of Chr7:19118311-19118422, and the target region of the SPN gene is selected from the positive chain of Chr16:29664517-29664618; the methylation sites in the target region of the Twist1 gene are selected from at least one of the following: Chr7:1911831 6. Chr7:19118321, Chr7:19118379, Chr7:19118403, Chr7:19118408 and Chr7:19118419; the methylation site of the target region of the SPN gene is selected from at least one of the following: Chr16:29664525, Chr16:29664538, Chr16:29664575, Chr16:29664578, Chr16:29664607 and Chr16:29664614.

[0053] The inventors of the present disclosure have discovered that the methylation levels of the methylation sites in the Twist1 gene target region and the SPN gene target region are significantly correlated with the incidence of bladder cancer. By detecting the methylation levels of the above-mentioned methylation sites, the purpose of accurately diagnosing bladder cancer can be achieved, with the advantages of high accuracy, strong sensitivity and strong specificity.

[0054] Primer sets, probe sets, and kits

[0055] In yet another aspect of the present disclosure, a primer set is provided. According to an embodiment of the present disclosure, the primer set is used to detect the aforementioned biomarkers, and the primer set includes: a first primer set, wherein the nucleotide sequences of the primers in the first primer set are shown in SEQ ID NOs: 1 and 2, respectively; and a second primer set, wherein the nucleotide sequences of the primers in the second primer set are shown in SEQ ID NOs: 4 and 5, respectively.

[0056] The first primer set can specifically identify and bind to the methylation sites in the aforementioned Twist1 gene target region, and the second primer set can specifically identify and bind to the methylation sites in the aforementioned SPN gene target region. Furthermore, by detecting the methylation levels of the methylation sites, the purpose of accurately diagnosing bladder cancer can be achieved.

[0057] In yet another aspect, the present disclosure provides a probe set. According to an embodiment of the present disclosure, the probe set is used to detect the aforementioned biomarkers. The probe set includes a first probe having a nucleotide sequence as shown in SEQ ID NO: 3; and a second probe having a nucleotide sequence as shown in SEQ ID NO: 6. Thus, the probe set disclosed herein can be used to detect the Twist1 gene and the SPN gene by fluorescent quantitative PCR, thereby diagnosing bladder cancer.

[0058] According to the embodiments of the present disclosure, the first probe is labeled with a FAM fluorescent reporter group at its 5' end and an MGB fluorescent quencher group at its 3' end; the second probe is labeled with a ROX fluorescent reporter group at its 5' end and an MGB fluorescent quencher group at its 3' end. Labeling different probes with different fluorescent groups facilitates multi-gene testing in the same reaction system, improving detection efficiency and reducing testing costs.

[0059] In yet another aspect, the present disclosure provides a kit. According to embodiments of the present disclosure, the kit is used to detect the aforementioned biomarkers, and includes at least one of the following: the aforementioned primer set and the aforementioned probe set. Thus, the kit can be used to detect methylation levels of the Twist1 and SPN genes, thereby facilitating the diagnosis of bladder cancer.

[0060] According to an embodiment of the present disclosure, the kit further includes at least one of the following: a sample collection container, a DNA extraction reagent, a methylation conversion reagent, a fluorescent quantitative PCR detection reagent, a primer for detecting the β-actin gene, and a probe for detecting the β-actin gene.

[0061] According to an embodiment of the present disclosure, the 5' end of the probe for detecting the β-actin gene is labeled with a VIC fluorescent reporter group, and the 3' end is labeled with an MGB fluorescent quencher group.

[0062] The sample collection container is used to collect a sample, which can be urine, sweat, blood, whole blood, serum, etc., preferably urine. The sample collection container can have a capacity of 1-10 mL or 1-3 mL. The disclosed kit enables accurate testing of small amounts of sample, thereby reducing the difficulty of sample collection and facilitating testing. There are no strict restrictions on the material and shape of the sample collection container, and the material and shape can be flexibly selected based on conventional practices in the field.

[0063] The DNA extraction reagent is used to extract DNA, and specifically can be a conventional DNA extraction reagent in the art.

[0064] Methylation conversion reagents can convert cytosine (C) in nucleic acid fragments into uracil (U), while 5-methylcytosine (5mC) remains unchanged. Furthermore, by designing primers specific to the methylation sequence, PCR amplification of the methylation site is performed. If an amplified fragment is obtained, it indicates that methylation is present at the site of the nucleic acid fragment. If no amplified fragment is obtained, it indicates that methylation is not present at the site of the nucleic acid fragment, thereby diagnosing bladder cancer. Specifically, the methylation conversion reagent can be a sulfite conversion reagent.

[0065] Fluorescence quantitative PCR detection reagents can include PCR buffer, dNTPs, DNA polymerase, and other commonly used reagents for fluorescence quantitative PCR. Primers and probes for detecting the β-actin gene can contain sequences commonly used in the art and are used to detect the expression level of a β-actin control gene. The 5' end of the probe for detecting the β-actin gene is labeled with a VIC fluorescent reporter group, enabling multiplex single-tube detection.

[0066] In addition, the present disclosure provides a method for using the aforementioned kit, comprising the following steps:

[0067] (1) Extracting genomic DNA from the biological sample to be tested;

[0068] (2) performing sulfite conversion on the genomic DNA of the biological sample to be tested;

[0069] (3) using the primer set and probe set in the aforementioned kit to perform methylation quantitative PCR detection on the sulfite-converted DNA;

[0070] (4) Analyze the test results.

[0071] application

[0072] In yet another aspect, the present disclosure provides the use of reagents for detecting the aforementioned biomarkers in the preparation of detection products. According to embodiments of the present disclosure, the detection products are used to diagnose bladder cancer. Thus, accurate diagnosis of bladder cancer can be achieved using biomarkers, primer sets, probe sets, or kits.

[0073] According to an embodiment of the present disclosure, the reagent for detecting a biomarker includes the aforementioned primer set and / or the aforementioned probe set.

[0074] According to an embodiment of the present disclosure, the detection product includes the aforementioned kit, reagent strip or chip.

[0075] It should be noted that the features and advantages described above for the biomarkers, primer sets, probe sets and kits are also applicable to this application and will not be repeated here.

[0076] method

[0077] In another aspect of the present disclosure, a method for detecting methylation levels of bladder cancer biomarkers is provided. According to an embodiment of the present disclosure, the method comprises detecting the aforementioned biomarkers in a biological sample using the aforementioned primer set and probe set. Thus, the disclosed method can accurately detect methylation levels of the Twist1 and SPN genes in bladder cancer. This method can be used for diagnosing bladder cancer, as well as for non-diagnostic purposes, such as studying the physiology or pathology of bladder cancer and screening for bladder cancer treatment drugs, thus possessing high application value.

[0078] According to an embodiment of the present disclosure, the method includes: detecting the aforementioned biomarkers in a biological sample using the aforementioned primer set and the aforementioned probe set.

[0079] It should be noted that the features and advantages described above for biomarkers, primer sets, and probe sets are also applicable to this method and will not be repeated here.

[0080] Device, system, electronic device, and computer-readable storage medium

[0081] In another aspect of the present disclosure, a device for diagnosing bladder cancer is provided. According to an embodiment of the present disclosure, the device for diagnosing bladder cancer includes: a detection unit adapted to detect methylation levels in the aforementioned biomarkers and obtain a detection result; and an analysis unit adapted to diagnose bladder cancer based on the detection result. As such, the device of the present disclosure offers simple and rapid operation, high detection accuracy, low cost, and convenient sampling, enabling non-invasive early diagnosis of bladder cancer and promising application prospects.

[0082] According to an embodiment of the present disclosure, the detection unit includes a detection device for detecting the methylation sites in the target region of the Twist1 gene and the methylation levels of the methylation sites in the target region of the SPN gene, including a fluorescence quantitative PCR instrument.

[0083] According to an embodiment of the present disclosure, the detection unit further comprises a DNA extraction device, which is used to extract DNA from a biological sample and includes a centrifuge, a mixer, and the like.

[0084] According to an embodiment of the present disclosure, the analysis unit is suitable for diagnosing bladder cancer based on the following judgment method: under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated positive; if Ct(Twist1)-Ct(β-actin)>9.4 or there is no amplification curve in the Twist1 gene target region, it is judged that the Twist1 gene is methylated negative; under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated negative. If the amplification curve of the target region of the SPN gene is S-shaped and Ct(β-actin) ≤ 35, and Ct(SPN)-Ct(β-actin) ≤ 10.1, the SPN gene is considered positive for methylation. If Ct(SPN)-Ct(β-actin) > 10.1 or there is no amplification curve in the target region of the SPN gene, the SPN gene is considered negative for methylation. If at least one of the Twist1 gene and the SPN gene is positive for methylation, the patient is diagnosed with bladder cancer. If both the Twist1 gene and the SPN gene are negative for methylation, the patient is diagnosed with bladder cancer. Specifically, the Ct value can be obtained using the software supporting the fluorescent quantitative PCR instrument.

[0085] In yet another aspect, the present disclosure provides an analysis system for diagnosing bladder cancer. According to an embodiment of the present disclosure, the analysis system for diagnosing bladder cancer includes: a data acquisition module configured to obtain detection results of the aforementioned biomarker methylation levels in a biological sample; and an analysis module configured to diagnose bladder cancer based on the detection results. Thus, the analysis system of the present disclosure can accurately diagnose bladder cancer.

[0086] According to an embodiment of the present disclosure, the analysis module is configured to diagnose bladder cancer based on the following judgment method: under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated positive; if Ct(Twist1)-Ct(β-actin)>9.4 or there is no amplification curve in the Twist1 gene target region, it is judged that the Twist1 gene is methylated negative; under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated negative. Under the premise that the amplification curve is S-shaped and Ct(β-actin)≤35, if the amplification curve of the target region of the SPN gene is S-shaped and Ct(SPN)-Ct(β-actin)≤10.1, the SPN gene is judged to be positive for methylation; if Ct(SPN)-Ct(β-actin)>10.1 or there is no amplification curve in the target region of the SPN gene, the SPN gene is judged to be negative for methylation; when at least one of the Twist1 gene and the SPN gene is positive for methylation, the patient is judged to have bladder cancer; when both the Twist1 gene and the SPN gene are negative for methylation, the patient is judged to not have bladder cancer.

[0087] In another aspect of the present disclosure, an electronic device is provided. According to an embodiment of the present disclosure, the electronic device includes a memory and a processor. The memory stores a program executable on the processor. When executed by the processor, the program diagnoses bladder cancer based on the methylation levels of the aforementioned biomarkers in a biological sample. Thus, the electronic device of the present disclosure can accurately diagnose bladder cancer. Specifically, the electronic device can be any intelligent terminal, including a fluorescent quantitative PCR instrument, a computer, a tablet computer, a computing cluster, or the like.

[0088] According to an embodiment of the present disclosure, when the program is executed by the processor, bladder cancer is diagnosed based on the following judgment method: under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated positive; if Ct(Twist1)-Ct(β-actin)>9.4 or there is no amplification curve in the Twist1 gene target region, it is judged that the Twist1 gene is methylated negative; under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated negative. Under the premise that the amplification curve is S-shaped and Ct(β-actin)≤35, if the amplification curve of the target region of the SPN gene is S-shaped and Ct(SPN)-Ct(β-actin)≤10.1, the SPN gene is judged to be positive for methylation; if Ct(SPN)-Ct(β-actin)>10.1 or there is no amplification curve in the target region of the SPN gene, the SPN gene is judged to be negative for methylation; when at least one of the Twist1 gene and the SPN gene is positive for methylation, the patient is judged to have bladder cancer; when both the Twist1 gene and the SPN gene are negative for methylation, the patient is judged to not have bladder cancer.

[0089] As used herein, the term "memory" refers to any computer program product, device, and / or system (e.g., a disk, an optical disk, a memory, a programmable logic system (PLD)) for providing machine instructions and / or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. The memory can be implemented in the form of a read-only memory (ROM), a static storage device, a dynamic storage device, or a random access memory (RAM). The memory can store operating devices and other applications. When the technical solutions provided in the embodiments of this specification are implemented by software or firmware, the relevant program code is stored in the memory and is called by the processor to execute the embodiment of the present application for diagnosing bladder cancer based on the methylation level of biomarkers in a biological sample.

[0090] In the present disclosure, the processor can be a general-purpose processor or a dedicated processor, etc. For example, it can be a baseband processor or a central processing unit. The baseband processor can be used to process communication protocols and communication data, and the central processing unit can be used to control communication equipment (such as base stations, baseband chips, terminal equipment, terminal equipment chips, DU or CU, etc.), execute computer programs, and process computer program data. The processor can be implemented in an integrated circuit (IC), an analog IC, a radio frequency integrated circuit RFIC, a mixed signal IC, an application specific integrated circuit (ASIC), a printed circuit board (PCB), an electronic device, etc. The processor and transceiver can also be manufactured using various IC process technologies, such as complementary metal oxide semiconductor (CMOS), N-type metal oxide semiconductor (nMetal-oxide-semiconductor, NMOS), P-type metal oxide semiconductor (positive channel metal oxide semiconductor, PMOS), bipolar junction transistor (bipolar junction transistor, BJT), bipolar CMOS (BiCMOS), silicon germanium (SiGe), gallium arsenide (GaAs), etc.

[0091] In another aspect, the present disclosure provides a computer-readable storage medium. According to an embodiment of the present disclosure, the computer-readable storage medium stores one or more programs, which can be executed by one or more processors to enable diagnosis of bladder cancer based on the methylation levels of the aforementioned biomarkers in a biological sample. Thus, the computer-readable storage medium of the present disclosure can accurately diagnose bladder cancer.

[0092] According to an embodiment of the present disclosure, when one or more programs are executed by one or more processors, bladder cancer is diagnosed based on the following judgment method: under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated positive; if Ct(Twist1)-Ct(β-actin)>9.4 or there is no amplification curve in the Twist1 gene target region, it is judged that the Twist1 gene is methylated negative; under the premise that the β-actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the Twist1 gene target region amplification curve is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated negative. Under the premise that the actin gene amplification curve is S-shaped and Ct(β-actin)≤35, if the amplification curve of the SPN gene target region is S-shaped and Ct(SPN)-Ct(β-actin)≤10.1, the SPN gene is judged to be methylation-positive; if Ct(SPN)-Ct(β-actin)>10.1 or there is no amplification curve in the SPN gene target region, the SPN gene is judged to be methylation-negative; when at least one of the Twist1 gene and the SPN gene is methylation-positive, the patient is judged to have bladder cancer; when both the Twist1 gene and the SPN gene are methylation-negative, the patient is judged to not have bladder cancer.

[0093] In this document, a computer-readable storage medium can be any available medium that can be accessed by a computer, or a data storage device such as a server or data center that includes one or more available media. Available media can include magnetic media (e.g., floppy disks, hard disks, magnetic tapes), optical media (e.g., high-density digital video discs (DVDs)), or semiconductor media (e.g., solid-state drives (SSDs)).

[0094] It should be noted that the features and advantages described above for the biomarkers, primer sets, and probe sets are also applicable to the device, system, electronic device, and computer-readable storage medium, and will not be repeated here.

[0095] Example 1

[0096] 1. Urine sample collection

[0097] Urine samples were collected from 38 patients with bladder cancer confirmed by pathological examination and 37 patients without bladder cancer undergoing routine physical examinations in a hospital in Shanghai. The collection volume of each urine sample was 10 mL. The collection process of all samples was approved by the ethics committee, and all volunteers signed informed consent forms. All samples were anonymized.

[0098] 2. DNA extraction from urine samples

[0099] Urine DNA was extracted using the nucleic acid extraction reagent (Shanghai Ruiyi Biotechnology Co., Ltd., Shanghai Minjibei 20230565) as follows:

[0100] (1) Add 30 μL of lysis buffer C solution, 2 mL of urine, and 100 μL of lysis buffer B to a clean centrifuge tube in sequence. Place the tube in a constant temperature mixer at 60°C and 1200 rpm for 20 minutes. After incubation, place the tube in an ice bath for 5-10 minutes.

[0101] (2) During the incubation process, prepare the lysate / magnetic bead mixture according to the table below and mix well.

[0102] Table 1 Reaction system

[0103] (3) Add the lysate / magnetic bead mixture prepared in step (2) to the sample tube in step (1). Vortex for 1 minute, then mix by inverting or using a blender for 5-10 minutes to keep the magnetic beads suspended.

[0104] (4) Place the centrifuge tube on a magnetic rack and let it stand until the magnetic beads are adsorbed on the magnetic rack and the solution in the tube becomes clear. Then flip the centrifuge tube to rinse the remaining magnetic beads on the bottle cap. Let it stand for about 1 minute and then discard the solution.

[0105] (5) Add 1 mL of wash buffer A to the centrifuge tube (please check whether anhydrous ethanol has been added before use), shake and mix thoroughly, and then transfer the suspension to a new 1.5 mL centrifuge tube.

[0106] (6) Place the centrifuge tube on a magnetic rack and let it stand for 1 minute, then discard the solution.

[0107] (7) Add 1 mL of wash buffer A to the centrifuge tube (please check whether anhydrous ethanol has been added before use), vortex for 5 seconds, and then place it on a thermomixer at 25°C and 1500 rpm for 2 minutes.

[0108] (8) Place the centrifuge tube on a magnetic stand and let it stand for 1 minute, then discard the solution.

[0109] (9) Add 1 mL of wash buffer B to the centrifuge tube (please check whether anhydrous ethanol has been added before use), vortex for 5 seconds, and then place it on a thermomixer at 25°C and 1500 rpm for 2 minutes to combine.

[0110] (10) Place the centrifuge tube on a magnetic stand and let it stand for 1 minute, then discard the solution.

[0111] (11) Repeat steps (9) and (10).

[0112] (12) After a brief centrifugation, the centrifuge tube was re-fixed on the magnetic stand and the solution at the bottom of the tube was removed with a pipette. The tube was opened and placed at room temperature for 5-10 minutes to allow the ethanol to fully evaporate (the surface of the magnetic beads became matte and there was no cracking of the beads when observed with the naked eye).

[0113] (13) Add 50 μL of sample eluent to the centrifuge tube and vortex to fully suspend the magnetic beads in the eluent. Then place the centrifuge tube on a constant temperature mixer at 25°C and 1500 rpm for 10 minutes.

[0114] (14) Fix the centrifuge tube on the magnetic stand and let it stand for 2 minutes. After the magnetic beads are completely adsorbed on the side wall of the centrifuge tube, use a pipette to transfer the eluate to a new centrifuge tube to obtain the DNA solution.

[0115] 3. Urine DNA sulfite conversion

[0116] The DNA extracted above was subjected to sulfite conversion using a Zymo Research kit (EZ-96 DNA Methylation-lightning MagPrep, Catalog No. D5047) to obtain sulfite-converted DNA, specifically comprising the following steps:

[0117] (1) Add 130 μL of CT Conversion Reagent to the 20 μL urine DNA solution extracted above, mix well, and centrifuge.

[0118] (2) Carry out the conversion reaction according to the conversion conditions in the following table:

[0119] Table 2 Reaction conditions

[0120] (3) First, add 600 μL of M-Binding Buffer and 10 μL of MagBinding (magnetic beads) into the centrifuge tube and mix by inverting.

[0121] (4) Transfer the transformed sample in step (2) to a centrifuge tube containing M-Binding Buffer and magnetic beads, shake and mix thoroughly, and incubate at room temperature for 5-10 minutes. During this period, invert or shake every 2-3 minutes to mix thoroughly to keep the magnetic beads in suspension.

[0122] (5) After incubation, centrifuge at low speed and place the centrifuge tube on a magnetic stand for magnetic separation for 3 minutes. Carefully remove the supernatant.

[0123] (6) Remove the centrifuge tube from the magnetic rack, add 400 μL M-Washing Buffer, vortex to resuspend the magnetic beads, centrifuge briefly at low speed, place the centrifuge tube on the magnetic rack for magnetic separation for 3 minutes, and carefully remove the supernatant.

[0124] (7) Remove the centrifuge tube from the magnetic stand, add 200 μL of L-Desulphonation Buffer, vortex to resuspend the magnetic beads, and incubate at room temperature for 20 min. During this period, shake or vortex every 5 min to keep the magnetic beads suspended.

[0125] (8) Turn on the constant temperature shaking incubator, set the temperature to 55°C, and do not shake.

[0126] (9) After the incubation in step (7) is completed, centrifuge the tube briefly and place it on a magnetic stand for magnetic separation for 3 minutes. Carefully remove the supernatant.

[0127] (10) Remove the centrifuge tube, add 400 μL M-Washing Buffer, vortex to resuspend the magnetic beads, centrifuge briefly at low speed, place the centrifuge tube on a magnetic stand for magnetic separation for 3 minutes, and carefully remove the supernatant.

[0128] (11) Repeat step (10) once.

[0129] (12) After discarding the supernatant, centrifuge again and place the centrifuge tube on a magnetic rack to remove as much residual liquid as possible without sucking up the magnetic beads.

[0130] (13) After opening the centrifuge tube, place it in a 55°C constant temperature shaking incubator to dry. The drying standard is that the color of the magnetic beads can be observed to change from bright black to reddish brown.

[0131] (14) After drying, add 50 μL of elution buffer, resuspend the magnetic beads, and incubate in a 55°C constant temperature shaking incubator at 1500 rpm for 4 min. After completion, place the beads on a magnetic stand for magnetic separation, and transfer the supernatant containing DNA to a new centrifuge tube to obtain sulfite-converted DNA for subsequent detection.

[0132] 4. qPCR detection

[0133] (1) Primer and probe information

[0134] The sequences of the Twist1 gene detection primers and probes, the SPN gene detection primers and probes, and the control β-actin gene detection primers and probes are shown in Table 3. The Twist1 gene detection primers and probes can be used to specifically amplify the methylation sites in the target region of the Twist1 gene, where the target region is based on the GRCh38.p14 reference genome, Chr7:19118311-19118422 negative strand, and the methylation sites are Chr7:19118316, Chr7:19118321, Chr7:19118379, Chr7:19118403, Chr7:19118408, and Chr7:19118419. SPN gene detection primers and probes can be used to specifically amplify the methylation sites of the target region of the SPN gene, where the target region is based on the GRCh38.p14 reference genome, Chr16:29664517-29664618 positive chain, and the methylation sites are Chr16:29664525, Chr16:29664538, Chr16:29664575, Chr16:29664578, Chr16:29664607 and Chr16:29664614.

[0135] Table 3 Primer and probe sequences

[0136] Note: The double-underlined CG positions in the above primer and probe sequences are the methylation sites detected for Twist1 and SPN genes.

[0137] (2) Methylation quantitative PCR was performed on the above-mentioned sulfite-converted DNA using primers and probes (Table 3).

[0138] 1) In this embodiment, multiplex detection of the Twist1 gene, SPN gene, and β-actin gene of the same sample is performed in the same tube. PCR primer probe premixes for the three genes can be prepared first. The PCR primer probe premix includes: 0.1 μM Twist1 gene detection probe, 0.3 μM Twist1 gene forward primer, 0.3 μM Twist1 gene reverse primer, 0.1 μM SPN gene detection probe, 0.3 μM SPN gene forward primer, 0.3 μM SPN gene reverse primer, 0.1 μM β-actin gene detection probe, 0.3 μM β-actin gene forward primer, 0.3 μM β-actin gene reverse primer, and deionized water is added to 2.5 μL.

[0139] 2) The PCR reaction system is shown in Table 4.

[0140] Table 4 PCR reaction system

[0141] 3) The PCR amplification procedure is shown in Table 5.

[0142] Table 5 PCR amplification program

[0143] 4) The prepared reaction system is amplified using an ABI7500 instrument.

[0144] 5) The analysis and judgment criteria of the test results are:

[0145] a. Baseline and threshold line adjustment:

[0146] Adjust the baseline for each gene individually, set the fluorescence value 1 to 2 cycles before the minimum Ct value of the sample in a PCR as the baseline value, set the threshold at the inflection point of the S-shaped amplification curve, and the instrument will automatically calculate the Ct value of each gene in the sample.

[0147] b. Sample validity confirmation:

[0148] ① The VIC channel amplification curve of the control gene β-actin is S-shaped and the Ct value is ≤35, indicating that the sample is valid;

[0149] ② If the VIC channel amplification curve of the control gene β-actin is S-shaped, the Ct value is greater than 35, or there is no amplification curve, the sample is invalid.

[0150] c. Confirmation of target gene methylation:

[0151] ① If the FAM channel amplification curve of the Twist1 gene is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged as positive for Twist1 gene methylation; if Ct(Twist1)-Ct(β-actin)>9.4 or there is no amplification curve, it is judged as negative for Twist1 gene methylation;

[0152] ②If the amplification curve of the ROX channel of the SPN gene is S-shaped and Ct(SPN)-Ct(β-actin)≤10.1, it is judged as positive SPN gene methylation; if Ct(SPN)-Ct(β-actin)>10.1 or there is no amplification curve, it is judged as negative SPN gene methylation.

[0153] 6) Under the premise that the sample is valid, interpret its test results:

[0154] ① When Twist1 gene methylation is positive and SPN gene methylation is also positive, the sample test result is positive;

[0155] ② When Twist1 gene methylation is positive and SPN gene methylation is negative, or Twist1 gene methylation is negative and SPN gene methylation is positive, the sample test result is positive;

[0156] ③When the Twist1 gene methylation is negative and the SPN gene methylation is also negative, the sample test result is negative.

[0157] 5. Sample test results

[0158] (1) A total of 75 samples were tested and analyzed using Twist1 gene, SPN gene alone as biomarkers, and Twist1 and SPN genes combined as biomarkers.

[0159] (2) The test results are shown in Figure 1, Figure 2, and Table 6. When the Twist1 gene is used alone as a biomarker, its sensitivity for detecting bladder cancer is 86.8%, its specificity is 94.6%, and its area under the receiver operating characteristic curve is 0.940. When the SPN gene is used alone as a biomarker, its sensitivity for detecting bladder cancer is 81.6%, its specificity is 94.6%, and its area under the receiver operating characteristic curve is 0.889. When the Twist1 and SPN genes are used together as biomarkers, their sensitivity for detecting bladder cancer is 92.1%, and their specificity is 94.6%.

[0160] Note: Sensitivity (true positive rate, sensitivity) = number of true positives / (number of true positives + number of false negatives)*100%.

[0161] It refers to the degree of correct judgment of patients, that is, the percentage of patients who are actually sick but are correctly diagnosed.

[0162] Specificity (true negative rate, specificity) = number of true negatives / (number of true negatives + number of false positives)*100%.

[0163] It refers to the degree of correct judgment of non-patients, that is, the percentage of people who are actually healthy but are correctly diagnosed as healthy.

[0164] From the above results, it can be concluded that using the combined detection of Twist1 and SPN genes as biomarkers can significantly improve the detection rate of bladder cancer, so the dual-gene combination has more advantages than single-gene detection.

[0165] The present invention has high sensitivity and strong specificity. Even when testing with a small sample volume of 2 mL, high levels of sensitivity and specificity can still be achieved. In addition, the detection method is simple, fast, low-cost, and non-invasive, so it has great application value in the field of molecular biology.

[0166] Table 6 Test results of 75 samples Note: "-" indicates that the VIC channel amplification curve of the control gene β-actin is S-shaped and the Ct value is ≤35, indicating that the sample is valid. However, there is no amplification curve for the target gene, so the Ct value calculation is not performed, and the methylation test result of the target gene is negative.

[0167] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A primer set, characterized in that: The primer set is used to detect biomarkers, and the biomarkers include methylation sites in the target region of the Twist1 gene and methylation sites in the target region of the SPN gene; Taking GRCh38.p14 as the reference genome, the Twist1 gene target region was selected from the negative strand of Chr7:19118311-19118422, and the SPN gene target region was selected from the positive strand of Chr16:29664517-29664618; The primer set includes: a first primer set, wherein the nucleotide sequences of the primers in the first primer set are shown in SEQ ID NOs: 1 and 2, respectively; The second primer set includes primers whose nucleotide sequences are shown in SEQ ID NOs: 4 and 5, respectively.

2. The primer set according to claim 1, characterized in that The methylation site of the target region of the Twist1 gene is selected from at least one of the following: Chr7:19118316, Chr7:19118321, Chr7:19118379, Chr7:19118403, Chr7:19118408 and Chr7:19118419; The methylation site of the SPN gene target region is selected from at least one of the following: Chr16:29664525, Chr16:29664538, Chr16:29664575, Chr16:29664578, Chr16:29664607 and Chr16:29664614.

3. A probe set, characterized in that: The probe set is used to detect biomarkers, and the biomarkers include methylation sites in the target region of the Twist1 gene and methylation sites in the target region of the SPN gene; Taking GRCh38.p14 as the reference genome, the Twist1 gene target region was selected from the negative strand of Chr7:19118311-19118422, and the SPN gene target region was selected from the positive strand of Chr16:29664517-29664618; The probe set includes: A first probe, the nucleotide sequence of which is shown in SEQ ID NO: 3; The second probe has a nucleotide sequence as shown in SEQ ID NO:

6.

4. The probe set according to claim 3, characterized in that The methylation site of the target region of the Twist1 gene is selected from at least one of the following: Chr7:19118316, Chr7:19118321, Chr7:19118379, Chr7:19118403, Chr7:19118408 and Chr7:19118419; The methylation site of the SPN gene target region is selected from at least one of the following: Chr16:29664525, Chr16:29664538, Chr16:29664575, Chr16:29664578, Chr16:29664607 and Chr16:29664614.

5. The probe set according to claim 3, characterized in that The 5' end of the first probe is labeled with a FAM fluorescent reporter group, and the 3' end is labeled with an MGB fluorescent quencher group; The 5' end of the second probe is labeled with a ROX fluorescent reporter group, and the 3' end is labeled with an MGB fluorescent quencher group.

6. A kit, characterized in that The kit is used to detect biomarkers, and the biomarkers include methylation sites in the target region of the Twist1 gene and methylation sites in the target region of the SPN gene; Taking GRCh38.p14 as the reference genome, the Twist1 gene target region was selected from the negative strand of Chr7:19118311-19118422, and the SPN gene target region was selected from the positive strand of Chr16:29664517-29664618; The kit comprises: the primer set according to claim 1 or 2 and the probe set according to any one of claims 3 to 5.

7. The kit according to claim 6, characterized in that The methylation site of the target region of the Twist1 gene is selected from at least one of the following: Chr7:19118316, Chr7:19118321, Chr7:19118379, Chr7:19118403, Chr7:19118408 and Chr7:19118419; The methylation site of the SPN gene target region is selected from at least one of the following: Chr16:29664525, Chr16:29664538, Chr16:29664575, Chr16:29664578, Chr16:29664607 and Chr16:29664614.

8. The kit according to claim 6, characterized in that The kit further comprises at least one of the following: a sample collection container, a DNA extraction reagent, a methylation conversion reagent, a fluorescent quantitative PCR detection reagent, a primer for detecting the β-actin gene, and a probe for detecting the β-actin gene.

9. The kit according to claim 8, characterized in that The sample collection container has a capacity of 1 to 10 mL; The 5' end of the probe for detecting the β-actin gene is labeled with a VIC fluorescent reporter group, and the 3' end is labeled with an MGB fluorescent quencher group.

10. Use of a reagent for detecting biomarkers in preparing a detection product, characterized in that: The detection product is used to diagnose bladder cancer; The biomarkers include methylation sites in the Twist1 gene target region and methylation sites in the SPN gene target region; Taking GRCh38.p14 as the reference genome, the target region of the Twist1 gene was selected from the negative chain of Chr7: 19118311-19118422, and the target region of the SPN gene was selected from the positive chain of Chr16: 29664517-29664618.

11. The use according to claim 10, characterized in that The methylation site of the target region of the Twist1 gene is selected from at least one of the following: Chr7:19118316, Chr7:19118321, Chr7:19118379, Chr7:19118403, Chr7:19118408 and Chr7:19118419; The methylation site of the SPN gene target region is selected from at least one of the following: Chr16:29664525, Chr16:29664538, Chr16:29664575, Chr16:29664578, Chr16:29664607 and Chr16:29664614.

12. The use according to claim 10, characterized in that The reagent for detecting biomarkers comprises the primer set according to claim 1 or 2 and / or the probe set according to any one of claims 3 to 5; The detection product includes a test kit, a reagent strip or a chip; The kit is selected from the kit according to any one of claims 6 to 9.

13. A diagnostic method for detecting methylation levels of bladder cancer biomarkers, characterized in that: The diagnostic method comprises: Detecting a biomarker in a biological sample using the primer set of claim 1 or 2 and the probe set of any one of claims 3 to 5; The biomarkers include methylation sites in the Twist1 gene target region and methylation sites in the SPN gene target region; Taking GRCh38.p14 as the reference genome, the target region of the Twist1 gene was selected from the negative chain of Chr7: 19118311-19118422, and the target region of the SPN gene was selected from the positive chain of Chr16: 29664517-29664618.

14. The method according to claim 13, characterized in that The methylation site of the target region of the Twist1 gene is selected from at least one of the following: Chr7:19118316, Chr7:19118321, Chr7:19118379, Chr7:19118403, Chr7:19118408 and Chr7:19118419; The methylation site of the SPN gene target region is selected from at least one of the following: Chr16:29664525, Chr16:29664538, Chr16:29664575, Chr16:29664578, Chr16:29664607 and Chr16:29664614.

15. A device for diagnosing bladder cancer, characterized in that: The device for diagnosing bladder cancer comprises: a detection unit, wherein the detection unit is suitable for detecting the methylation level in the biomarker and obtaining a detection result; an analyzing unit adapted to diagnose bladder cancer based on the detection result; The biomarkers include methylation sites in the Twist1 gene target region and methylation sites in the SPN gene target region; Taking GRCh38.p14 as the reference genome, the target region of the Twist1 gene was selected from the negative chain of Chr7: 19118311-19118422, and the target region of the SPN gene was selected from the positive chain of Chr16: 29664517-29664618.

16. The device according to claim 15, characterized in that The methylation site of the target region of the Twist1 gene is selected from at least one of the following: Chr7:19118316, Chr7:19118321, Chr7:19118379, Chr7:19118403, Chr7:19118408 and Chr7:19118419; The methylation site of the SPN gene target region is selected from at least one of the following: Chr16:29664525, Chr16:29664538, Chr16:29664575, Chr16:29664578, Chr16:29664607 and Chr16:29664614.

17. The device according to claim 15, characterized in that The detection unit contains a detection device; The detection device includes a fluorescence quantitative PCR instrument.

18. The device according to claim 15, characterized in that The analyzing unit is adapted to diagnose bladder cancer based on the following determinations: Under the premise that the amplification curve of the β-actin gene is S-shaped and Ct(β-actin)≤35, if the amplification curve of the target region of the Twist1 gene is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated positive; if Ct(Twist1)-Ct(β-actin)>9.4 or there is no amplification curve of the target region of the Twist1 gene, it is judged that the Twist1 gene is methylated negative; On the premise that the amplification curve of the β-actin gene is S-shaped and Ct(β-actin)≤35, if the amplification curve of the target region of the SPN gene is S-shaped and Ct(SPN)-Ct(β-actin)≤10.1, it is judged as positive for SPN gene methylation; if Ct(SPN)-Ct(β-actin)>10.1 or there is no amplification curve in the target region of the SPN gene, it is judged as negative for SPN gene methylation; When at least one of the Twist1 gene and the SPN gene is methylation-positive, the patient is diagnosed with bladder cancer; When both Twist1 gene and SPN gene are methylation negative, it is determined that there is no bladder cancer; The detection unit further comprises a DNA extraction device.

19. An analysis system for diagnosing bladder cancer, characterized in that The analysis system for diagnosing bladder cancer comprises: a data acquisition module, the data acquisition module being configured to acquire a detection result of a biomarker methylation level in a biological sample; an analyzing module configured to diagnose bladder cancer based on the detection result; The biomarkers include methylation sites in the Twist1 gene target region and methylation sites in the SPN gene target region; Taking GRCh38.p14 as the reference genome, the target region of the Twist1 gene was selected from the negative chain of Chr7: 19118311-19118422, and the target region of the SPN gene was selected from the positive chain of Chr16: 29664517-29664618.

20. The analysis system according to claim 19, characterized in that The methylation site of the target region of the Twist1 gene is selected from at least one of the following: Chr7:19118316, Chr7:19118321, Chr7:19118379, Chr7:19118403, Chr7:19118408 and Chr7:19118419; The methylation site of the SPN gene target region is selected from at least one of the following: Chr16:29664525, Chr16:29664538, Chr16:29664575, Chr16:29664578, Chr16:29664607 and Chr16:29664614.

21. The analysis system according to claim 19, characterized in that The analysis module is configured to diagnose bladder cancer based on the following determinations: Under the premise that the amplification curve of the β-actin gene is S-shaped and Ct(β-actin)≤35, if the amplification curve of the target region of the Twist1 gene is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated positive; if Ct(Twist1)-Ct(β-actin)>9.4 or there is no amplification curve of the target region of the Twist1 gene, it is judged that the Twist1 gene is methylated negative; On the premise that the amplification curve of the β-actin gene is S-shaped and Ct(β-actin)≤35, if the amplification curve of the target region of the SPN gene is S-shaped and Ct(SPN)-Ct(β-actin)≤10.1, it is judged as positive for SPN gene methylation; if Ct(SPN)-Ct(β-actin)>10.1 or there is no amplification curve in the target region of the SPN gene, it is judged as negative for SPN gene methylation; When at least one of the Twist1 gene and the SPN gene is methylation-positive, the patient is diagnosed with bladder cancer; When both the Twist1 gene and the SPN gene are methylation-negative, it is determined that the patient does not have bladder cancer.

22. An electronic device, characterized in that: The electronic device includes a memory and a processor, wherein the memory stores a program that can be run on the processor, and when the program is executed by the processor, it realizes the diagnosis of bladder cancer based on the methylation level of the biomarker in the biological sample; The biomarkers include methylation sites in the Twist1 gene target region and methylation sites in the SPN gene target region; Taking GRCh38.p14 as the reference genome, the target region of the Twist1 gene was selected from the negative chain of Chr7: 19118311-19118422, and the target region of the SPN gene was selected from the positive chain of Chr16: 29664517-29664618.

23. The electronic device according to claim 22, wherein: The methylation site of the target region of the Twist1 gene is selected from at least one of the following: Chr7:19118316, Chr7:19118321, Chr7:19118379, Chr7:19118403, Chr7:19118408 and Chr7:19118419; The methylation site of the SPN gene target region is selected from at least one of the following: Chr16:29664525, Chr16:29664538, Chr16:29664575, Chr16:29664578, Chr16:29664607 and Chr16:29664614.

24. The electronic device according to claim 22, characterized in that: When the program is executed by the processor, bladder cancer is diagnosed based on the following determination method: Under the premise that the amplification curve of the β-actin gene is S-shaped and Ct(β-actin)≤35, if the amplification curve of the target region of the Twist1 gene is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated positive; if Ct(Twist1)-Ct(β-actin)>9.4 or there is no amplification curve of the target region of the Twist1 gene, it is judged that the Twist1 gene is methylated negative; On the premise that the amplification curve of the β-actin gene is S-shaped and Ct(β-actin)≤35, if the amplification curve of the target region of the SPN gene is S-shaped and Ct(SPN)-Ct(β-actin)≤10.1, it is judged as positive for SPN gene methylation; if Ct(SPN)-Ct(β-actin)>10.1 or there is no amplification curve in the target region of the SPN gene, it is judged as negative for SPN gene methylation; When at least one of the Twist1 gene and the SPN gene is methylation-positive, the patient is diagnosed with bladder cancer; When both the Twist1 gene and the SPN gene are methylation-negative, it is determined that the patient does not have bladder cancer.

25. A computer-readable storage medium, characterized in that The computer-readable storage medium stores one or more programs, and the one or more programs can be executed by one or more processors to diagnose bladder cancer based on the methylation level of the biomarker in the biological sample; The biomarkers include methylation sites in the Twist1 gene target region and methylation sites in the SPN gene target region; Taking GRCh38.p14 as the reference genome, the target region of the Twist1 gene was selected from the negative chain of Chr7: 19118311-19118422, and the target region of the SPN gene was selected from the positive chain of Chr16: 29664517-29664618.

26. The computer-readable storage medium according to claim 25, wherein: The methylation site of the target region of the Twist1 gene is selected from at least one of the following: Chr7:19118316, Chr7:19118321, Chr7:19118379, Chr7:19118403, Chr7:19118408 and Chr7:19118419; The methylation site of the SPN gene target region is selected from at least one of the following: Chr16:29664525, Chr16:29664538, Chr16:29664575, Chr16:29664578, Chr16:29664607 and Chr16:29664614.

27. The computer-readable storage medium according to claim 25, wherein: When the one or more programs are executed by one or more processors, the diagnosis of bladder cancer is achieved based on the following judgment method: Under the premise that the amplification curve of the β-actin gene is S-shaped and Ct(β-actin)≤35, if the amplification curve of the target region of the Twist1 gene is S-shaped and Ct(Twist1)-Ct(β-actin)≤9.4, it is judged that the Twist1 gene is methylated positive; if Ct(Twist1)-Ct(β-actin)>9.4 or there is no amplification curve of the target region of the Twist1 gene, it is judged that the Twist1 gene is methylated negative; On the premise that the amplification curve of the β-actin gene is S-shaped and Ct(β-actin)≤35, if the amplification curve of the target region of the SPN gene is S-shaped and Ct(SPN)-Ct(β-actin)≤10.1, it is judged as positive for SPN gene methylation; if Ct(SPN)-Ct(β-actin)>10.1 or there is no amplification curve in the target region of the SPN gene, it is judged as negative for SPN gene methylation; When at least one of the Twist1 gene and the SPN gene is methylation-positive, the patient is diagnosed with bladder cancer; When both the Twist1 gene and the SPN gene are methylation-negative, it is determined that the patient does not have bladder cancer.

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