Primer set, probe set, kit, and method for detecting bladder cancer biomarkers

Abstract

A urine exosome marker panel for detecting bladder cancer and the use thereof. The provided biomarker panel comprising IGF2, KRT20, SPAG5, NRP1 and DHRS2 is used for detecting bladder cancer patients. The marker panel is used to construct a prediction model, and the model is trained by means of a training set and optimized by means of a test verification set, so as to finally obtain an early diagnosis prediction model for bladder cancer. The provided exosome marker panel and prediction model enable relatively accurate early screening of clinical samples from patients clinically suspected of having bladder cancer, presenting with symptoms such as hematuria, frequent urination, urgent urination, and dysuria, and for whom cystoscopy is recommended by clinical diagnosis, wherein the optimal marker panel is IGF2+KRT20+SPAG5+NRP1, with an AUC of 0.902, a sensitivity of 86.8%, a specificity of 83.0%, and an accuracy of 84.8%, demonstrating favorable clinical diagnostic value.

Description

Primer set, probe set, kit and method for detecting bladder cancer biomarker TECHNICAL FIELD

[0001] The present application belongs to the field of biomolecular detection, and particularly relates to a primer set, probe set, kit and method for detecting bladder cancer biomarker. BACKGROUND

[0002] Bladder cancer is a malignant tumor that occurs on the mucosa of the bladder, and is the most common malignant tumor of the urinary system and one of the ten most common tumors in the body. It accounts for the first place in the incidence of urogenital system tumors in China. The 5-year survival rate of early bladder cancer is 70%, the 5-year survival rate of medium bladder cancer is 38%, and the 5-year survival rate of advanced bladder cancer is 6%. Bladder cancer patients usually have poor prognosis, and the overall prognosis of bladder cancer patients in China is poor. The survival time of bladder cancer patients is closely related to the malignant tumor stage at the time of clinical diagnosis. Early detection of bladder cancer can increase the chance of surgery to retain the bladder and improve the overall survival rate of patients, and can also reduce the treatment cost of advanced bladder cancer. Non-invasive diagnosis can provide a convenient diagnosis for patients with recurrent bladder cancer, so auxiliary diagnosis and recurrence detection of high-risk groups of bladder cancer are effective means to improve the overall survival rate of bladder cancer patients in China.

[0003] Extracellular vesicles (EVs) are vesicles secreted by cells with a phospholipid bilayer structure, which can regulate the biological characteristics of recipient cells by carrying biological macromolecules, and participate in physiological and pathological processes of cells. Exosomes are an important subpopulation of EVs, which encapsulate proteins, mRNAs and microRNAs, etc. They are the medium of intercellular short-distance communication in health and disease, and participate in multiple processes such as tumor occurrence, development, invasion and metastasis. At the same time, compared with other body fluids, urine has the characteristics of safe sampling, large sample size and non-invasive, and research has found that urinary exosome RNA can be used as a marker to effectively detect urinary system cancer.

[0004] Currently, urine cytology, NMP22 protein and imaging are the main tools for screening bladder cancer, but all have inherent limitations. Urine cytology is used to detect malignant tumors of the urinary system, and the examination method is simple, non-invasive and high in specificity, but sometimes the positive rate is low and multiple examinations are required; specific protein markers such as urinary nuclear matrix protein NMP22 have higher sensitivity than urine cytology, but the performance cannot meet the clinical needs; imaging has no advantage in detecting early stage bladder cancer, and may have similar imaging performance with other types of bladder tumors, which is difficult to distinguish and requires cystoscopy and pathological biopsy for diagnosis. How to improve the early detection rate while avoiding over-diagnosis and treatment is a great challenge in early screening of bladder cancer. The development of a urine exosome-based liquid biopsy product for bladder cancer provides an achievable cancer screening path for bladder cancer patients. SUMMARY

[0005] To solve the problems in the prior art, the application provides a urine exosome marker combination and kit for early detection of bladder cancer, which obtains a urine sample by a non-invasive method, and the urine sample does not need to be collected in the morning or separated from urine exfoliated cells before collection. By jointly detecting bladder cancer-related gene expression, the tumor detection rate can be significantly improved, and the method has the characteristics of non-invasiveness, precision and rapidness.

[0006] Receiver operating characteristic curve (ROC curve): is a curve drawn according to a series of different binary classification methods (cut-off value or decision threshold), with sensitivity (true positive rate) as the vertical coordinate and 1-specificity (false positive rate) as the horizontal coordinate. The area under the ROC curve is an important test accuracy index, and the larger the area under the ROC curve (AUC), the greater the diagnostic value of the test.

[0007] To achieve the above object, the technical scheme adopted by the application comprises:

[0008] The first aspect of the application discloses a urine exosome marker combination for bladder cancer detection, wherein the marker combination comprises a cell proliferation regulatory factor and a keratin.

[0009] Preferably, the cell proliferation regulatory factor is selected from IGF2, and the keratin is selected from KRT20.

[0010] The marker combination further comprises one or more genes selected from SPAG5, NRP1, CCNB1, CDC20, IGFBP5, VEGFA and MDK.

[0011] As a preferred embodiment of the application, the marker combination comprises IGF2, KRT20, SPAG5 and NRP1.

[0012] wherein the marker combination further comprises a reference gene selected from DHRS2 or ABL1.

[0013] The second aspect of the present application discloses the use of the above marker combination in the preparation of a bladder cancer detection product.

[0014] wherein the product comprises a detection kit. The kit comprises detection reagents for detecting the above marker combination.

[0015] The detection reagents comprise primer probes for detecting the gene levels in the marker combination. The primer probe sequences of the marker combination are shown in SEQ ID NO: 1-27. The primer probe sequences of the reference gene are shown in SEQ ID NO: 28-33.

[0016] The primer probe combination comprises: primers for amplifying IGF2, the upstream primer sequence of which is shown in SEQ ID NO: 1, and the downstream primer sequence of which is shown in SEQ ID NO: 2; primers for amplifying KRT20, the upstream primer sequence of which is shown in SEQ ID NO: 4, and the downstream primer sequence of which is shown in SEQ ID NO: 5; primers for amplifying SPAG5, the upstream primer sequence of which is shown in SEQ ID NO: 7, and the downstream primer sequence of which is shown in SEQ ID NO: 8; primers for amplifying NRP1, the upstream primer sequence of which is shown in SEQ ID NO: 10, and the downstream primer sequence of which is shown in SEQ ID NO: 11; primers for amplifying CDC20, the upstream primer sequence of which is shown in SEQ ID NO: 13, and the downstream primer sequence of which is shown in SEQ ID NO: 14; primers for amplifying CCNB1, the upstream primer sequence of which is shown in SEQ ID NO: 16, and the downstream primer sequence of which is shown in SEQ ID NO: 17; primers for amplifying IGFBP5, the upstream primer sequence of which is shown in SEQ ID NO: 19, and the downstream primer sequence of which is shown in SEQ ID NO: 20; primers for amplifying VEGFA, the upstream primer sequence of which is shown in SEQ ID NO: 22, and the downstream primer sequence of which is shown in SEQ ID NO: 23; primers for amplifying MDK, the upstream primer sequence of which is shown in SEQ ID NO: 25, and the downstream primer sequence of which is shown in SEQ ID NO: 26.

[0017] Preferably, the primer probe combination further comprises: primers for amplifying the reference gene DHRS2, the upstream primer sequence of which is shown in SEQ ID NO: 28, and the downstream primer sequence of which is shown in SEQ ID NO: 29; primers for amplifying the reference gene ABL1, the upstream primer sequence of which is shown in SEQ ID NO: 31, and the downstream primer sequence of which is shown in SEQ ID NO: 32.

[0018] Preferably, the probe primer combination further comprises a detection probe, wherein the probe sequence for detecting IGF2 is shown as SEQ ID NO: 3, the probe sequence for detecting KRT20 is shown as SEQ ID NO: 6, the probe sequence for detecting SPAG5 is shown as SEQ ID NO: 9, the probe sequence for detecting NRP1 is shown as SEQ ID NO: 12, the probe sequence for detecting CDC20 is shown as SEQ ID NO: 15, the probe sequence for detecting CCNB1 is shown as SEQ ID NO: 18, the probe sequence for detecting IGFBP5 is shown as SEQ ID NO: 21, the probe sequence for detecting VEGFA is shown as SEQ ID NO: 24, the probe sequence for detecting MDK is shown as SEQ ID NO: 27, the probe sequence for detecting DHRS2 is shown as SEQ ID NO: 30, and the probe sequence for detecting ABL1 is shown as SEQ ID NO: 33. Comprising:

[0019] IGF2 upstream primer: 5'-CTCCAGTTCGTCTGTGGG-3' (SEQ ID NO: 1);

[0020] IGF2 downstream primer: 5'-AAACAGCACTCCTCAACGAT-3' (SEQ ID NO: 2);

[0021] IGF2 probe: 5'-CTTCTACTTCAGCAGGCCCGCAAGCCG-3' (SEQ ID NO: 3);

[0022] KRT20 upstream primer: 5'-TCCAGTCCCATCTCAGCATGAAAG-3' (SEQ ID NO: 4);

[0023] KRT20 downstream primer: 5'-TTGGCTAACTGGCTGCTGTAA-3' (SEQ ID NO: 5);

[0024] KRT20 probe: 5'-GGAGCACACTCTAGAGGAGACCAAGGCCCG-3' (SEQ ID NO: 6);

[0025] SPAG5 upstream primer: 5'-AAGCAAAGGCCAAAGCCAAG-3' (SEQ ID NO: 7);

[0026] SPAG5 downstream primer: 5'-CACTGGTGGGTCACATCTCG-3' (SEQ ID NO: 8);

[0027] SPAG5 probe: 5'-GACGCCAAGCCTGGATGCCAAGGAGCC-3' (SEQ ID NO: 9);

[0028] NRP1 upstream primer: 5'-TACCTATGCTGGTGCCAGTG-3' (SEQ ID NO: 10);

[0029] NRP1 downstream primer: 5'-GGGCTTGGAGAGGCAGTATC-3' (SEQ ID NO: 11);

[0030] NRP1 probe: 5'-GCCAGTGCCAGAGCCAGAACCTGAGCC-3' (SEQ ID NO: 12);

[0031] CDC20 upstream primer: 5'-GGATCTACCATACCCATTGACTA-3' (SEQ ID NO: 13);

[0032] CDC20 downstream primer: 5'-GGCTACCACTTGACCTGTA-3' SEQ ID NO: 14;

[0033] CDC20 probe: 5'-ACACAAAACTACAGGTCAAGTGGTAGCCA-3' SEQ ID NO: 15;

[0034] CCNB1 upstream primer: 5'-TGTCTTCAGGGCCATTTCTT-3' SEQ ID NO: 16,

[0035] CCNB1 downstream primer: 5'-AGGTGTTGTGTAGTTCTGGG-3' SEQ ID NO: 17,

[0036] CCNB1 probe: 5'-TCTCTGACTACGAAACACATGGTGCAGGA-3' SEQ ID NO: 18;

[0037] IGFBP5 upstream primer: 5'-GGGTTTGCCTCAACGAAAAGAG-3' SEQ ID NO: 19;

[0038] IGFBP5 downstream primer: 5'-TGTGTTTGGGCCGGAAGAT-3' SEQ ID NO: 20;

[0039] IGFBP5 probe: 5'-GCACGAGGAGCCCACCACCTCTGAGAT-3' SEQ ID NO: 21;

[0040] VEGFA upstream primer: 5'-GTCCAACATCACCATGCAGATTA-3' SEQ ID NO: 22;

[0041] VEGFA downstream primer: 5'-TGTTGTGCTGTAGGAAGCTCA-3' SEQ ID NO: 23;

[0042] VEGFA probe: 5'-TGCGGATCAAACCTCACCAAGGCCAGCA-3' SEQ ID NO: 24;

[0043] MDK upstream primer: 5'-TGGAAAAAGATCCCCCGCTTG-3' SEQ ID NO: 25;

[0044] MDK downstream primer: 5'-CACTGGAGATGAGTTGTTGCTG-3' SEQ ID NO: 26;

[0045] MDK probe: 5'-CCCCATCACTGTGCAAGCTGGGGCTGC-3' SEQ ID NO: 27;

[0046] DHRS2 upstream primer: 5'-ACCAGTGAGCAGATCTGGGA-3' (SEQ ID NO: 28);

[0047] DHRS2 downstream primer: 5'-TCCATGTAGGGCAGCAACTG-3' (SEQ ID NO: 29);

[0048] DHRS2 probe: 5'-ACGTGAAGTCCCCAGCCCTGCTGCT-3' (SEQ ID NO: 30);

[0049] ABL1 upstream primer: 5'-GCTTCTGATGGCAAGCTCTA-3' SEQ ID NO: 31,

[0050] ABL1 downstream primer: 5'-GGCCACCGTTGAATGATGATG-3' SEQ ID NO: 32;

[0051] ABL1 probe: 5'-AGAGCCGCTTCAACACCCTGGCCGAGT-3' SEQ ID NO: 33.

[0052] The probe is labeled with a fluorescent reporter group selected from FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3 or JOE; and a fluorescent quencher group selected from BHQ1 or BHQ2.

[0053] Preferably, the fluorescent group of the probe as shown in SEQ ID NO: 3 is HEX; the fluorescent group of the probe as shown in SEQ ID NO: 6 is ROX; the fluorescent group of the probe as shown in SEQ ID NO: 9 is CY5; and the fluorescent group of the probe as shown in SEQ ID NO: 12 is FAM. Further, the 3' end of the probe also has a quencher group, such as BHQ1, BHQ2, etc.

[0054] In a specific embodiment, the quencher group of the probe as shown in SEQ ID NO: 3 is BHQ1; in a specific embodiment, the quencher group of the probe as shown in SEQ ID NO: 6 and SEQ ID NO: 9 is BHQ2; and in a specific embodiment, the quencher group of the probe as shown in SEQ ID NO: 12 is BHQ1.

[0055] In a specific embodiment, the fluorescent group of the probe as shown in SEQ ID NO: 15 is HEX and the quencher group is BHQ1; the fluorescent group of the probe as shown in SEQ ID NO: 18 is FAM and the quencher group is BHQ1; the fluorescent group of the probe as shown in SEQ ID NO: 21 is HEX and the quencher group is BHQ1; the fluorescent group of the probe as shown in SEQ ID NO: 24 is CY5 and the quencher group is BHQ2; the fluorescent group of the probe as shown in SEQ ID NO: 27 is ROX and the quencher group is BHQ2; the fluorescent group of the probe as shown in SEQ ID NO: 30 is FAM and the quencher group is BHQ1; and the fluorescent group of the probe as shown in SEQ ID NO: 33 is ROX and the quencher group is BHQ2.

[0056] The detection is achieved by detecting exosomes in the urine of the patient.

[0057] Preferably, the kit further comprises reagents for extracting exosomes from urine; and the kit further comprises a positive control and a negative control.

[0058] As an embodiment of the present application, the kit of the present application comprises the following components: a RT-qPCR reaction solution containing primers and probes specifically recognizing the mRNA sequences of IGF2, KRT20, SPAG5, NRP1 and DHRS2, an enzyme mixture containing reverse transcriptase, DNA polymerase, UDG enzyme, positive quality control, and negative quality control. Further, the final concentration of the primers in the RT-qPCR reaction system is 0.2 μM-0.4 μM, preferably 0.4 μM; the final concentration of the probes in the RT-qPCR reaction system is 0.1 μM-0.3 μM, preferably 0.2 μM. The RT-qPCR reaction system further comprises Tris buffer, magnesium ions, dA / G / C / UTPs. The final concentration of the magnesium ions is 5 mM, the final concentration of the dATP is 0.4 mM, the final concentration of the dCTP is 0.4 mM, the final concentration of the dGTP is 0.4 mM, and the final concentration of the dUTP is 0.8 mM.

[0059] Further, the present application also discloses a bladder cancer prediction model based on a combination of urine exosome biomarkers. The model is used to convert the marker combination gene expression level into the bladder cancer risk level of the patient to be tested, and the algorithm of the model is:

[0060] Output value = {Ct(Target 1)-Ct(internal reference)} x a + {Ct(Target 2)-Ct(internal reference)} x b + {Ct(Target 3)-Ct(internal reference)} x c + {Ct(Target 4)-Ct(internal reference)} x d + … + {Ct(Target N)-Ct(internal reference)} x n + z;

[0061] In the formula, a, b, c…n are coefficients, z is a constant, the coefficients are between -1 and 1, Ct(internal reference) is the Ct value of the internal reference gene, and Ct(Target 1)…Ct(Target N) are the Ct values of each gene in the marker combination of claims 1-4.

[0062] The output value of the above algorithm is used to predict whether the patient is at low risk or high risk of bladder cancer. For example, patients with an output value calculated using the above algorithm less than 0.35 are identified as having a lower risk of bladder cancer, and patients with an output value equal to or higher than 0.35 are identified as having a higher risk of bladder cancer.

[0063] As a preferred embodiment of the present application, a kit for early diagnosis of bladder cancer is provided. The kit is used to detect the mRNA expression levels of IGF2, KRT20, SPAG5, NRP1 and DHRS2 in urine exosomes, and then calculate the output value using the above-mentioned bladder cancer prediction model and compare it with the cutoff value to analyze the risk of the subject suffering from bladder cancer.

[0064] The application further discloses a method for using the kit in early diagnosis of bladder cancer, which comprises the following steps:

[0065] urine samples of suspected bladder cancer patients are collected, starting from the first urine, and the volume is 30-50 mL;

[0066] exosomes are separated and purified from the urine samples;

[0067] RNA in the exosomes is extracted;

[0068] the primers and probe compositions and the kit of the IGF2, KRT20, SPAG5, NRP1 and DHRS2 genes are used for fluorescence quantitative RT-qPCR amplification of nucleic acids;

[0069] expression values of the biomarkers are obtained, and the risk of cancer of the subject is evaluated by using the bladder cancer prediction model of the application.

[0070] The expression values of the IGF2, KRT20, SPAG5, NRP1 and DHRS2 biomarker combination in the urine exosomes of bladder cancer patients have a statistically significant difference compared with the expression values in the control sample. The marker combination is used to construct a prediction model, the model is trained by a training set, and the model is optimized by a test validation set, and finally an early diagnosis prediction model for bladder cancer is obtained.

[0071] The exosome marker combination and the prediction model can be used for early screening of clinical samples of patients suspected of having bladder cancer, such as hematuria, frequent urination, urinary urgency and urinary pain, and clinical diagnosis suggests cystoscopy, wherein the best combination marker is IGF2+KRT20+SPAG5+NRP1, the AUC is 0.902, the sensitivity is 86.8%, the specificity is 83.0%, and the accuracy is 84.8%, which has good clinical diagnostic value.

[0072] The application collects urine samples non-invasively and detects exosomes thereof, analyzes the expression of bladder cancer-related mRNA in the exosomes, and establishes an early screening prediction model for bladder cancer by logistic regression. The established bladder cancer screening prediction model has the characteristics of high sensitivity and high specificity, and finally the model can be used for classification and screening of cancer and non-cancer samples. BRIEF DESCRIPTION OF DRAWINGS

[0073] FIG. 1 is a ROC curve of the training set with the true positive rate (sensitivity) as the vertical coordinate and the false positive rate (1-specificity) as the horizontal coordinate, and the AUC values of different exosome marker combinations in the training set.

[0074] a. The AUC of the marker combination IGF2 + KRT20 + SPAG5 was 0.896;

[0075] b. The AUC of the marker combination IGF2 + KRT20 + SPAG5 + MDK was 0.898;

[0076] c. The AUC of the marker combination IGF2 + KRT20 + SPAG5 + IGFBP5 was 0.904;

[0077] d. The AUC of the marker combination IGF2 + KRT20 + SPAG5 + VEGFA was 0.911 ;

[0078] e. The AUC of the marker combination IGF2 + KRT20 + SPAG5 + CCNB1 was 0.910;

[0079] f. The AUC of the marker combination IGF2 + KRT20 + SPAG5 + CDC20 was 0.920;

[0080] g. The AUC of the marker combination IGF2 + KRT20 + SPAG5 + NRP1 was 0.923.

[0081] Figure 2. ROC curves of the training set plotted with the true positive rate (sensitivity) as the vertical coordinate and the false positive rate (1 - specificity) as the horizontal coordinate. AUC values of the training set for different combinations of exosomal markers.

[0082] a. The AUC of the marker combination IGF2 + KRT20 + NRP1 was 0.890;

[0083] b. The AUC of the marker combination IGF2 + KRT20 + NRP1 + VEGFA was 0.891 ;

[0084] c. The AUC of the marker combination IGF2 + KRT20 + NRP1 + CCNB1 was 0.903;

[0085] d. The AUC of the marker combination IGF2 + KRT20 + NRP1 + CDC20 was 0.912;

[0086] e. The AUC of the marker combination IGF2 + KRT20 + NRP1 + IGFBP5 was 0.905;

[0087] f. The AUC of the marker combination IGF2 + KRT20 + NRP1 + MDK was 0.915.

[0088] Figure 3. ROC curves of the validation set plotted with the true positive rate (sensitivity) as the vertical coordinate and the false positive rate (1 - specificity) as the horizontal coordinate. AUC values of the validation set for different combinations of exosomal markers.

[0089] a. The AUC of the marker combination IGF2 + KRT20 + SPAG5 was 0.850;

[0090] b. The AUC of the marker combination IGF2 + KRT20 + SPAG5 + MDK was 0.858;

[0091] c. The AUC of the marker combination IGF2 + KRT20 + SPAG5 + IGFBP5 was 0.869;

[0092] d. The AUC of the marker combination IGF2 + KRT20 + SPAG5 + VEGFA was 0.877;

[0093] e. The AUC of the marker combination IGF2 + KRT20 + SPAG5 + CCNB1 was 0.876;

[0094] f. The AUC of the marker combination IGF2 + KRT20 + SPAG5 + CDC20 was 0.898;

[0095] g. The AUC of the marker combination IGF2 + KRT20 + SPAG5 + NRP1 was 0.902.

[0096] Figure 4. ROC curve of the validation set plotted with true positive rate (sensitivity) as the vertical coordinate and false positive rate (1 - specificity) as the horizontal coordinate. AUC values of different exosome marker combinations for the validation set.

[0097] a. The AUC of the marker combination IGF2 + KRT20 + NRP1 was 0.870;

[0098] b. The AUC of the marker combination IGF2 + KRT20 + NRP1 + VEGFA was 0.875;

[0099] c. The AUC of the marker combination IGF2 + KRT20 + NRP1 + CCNB1 was 0.876;

[0100] d. The AUC of the marker combination IGF2 + KRT20 + NRP1 + CDC20 was 0.883;

[0101] e. The AUC of the marker combination IGF2 + KRT20 + NRP1 + IGFBP5 was 0.897;

[0102] f. The AUC of the marker combination IGF2 + KRT20 + NRP1 + MDK was 0.909.

[0103] Figure 5. ROC curve of the validation set with true positive rate (sensitivity) as the vertical coordinate and false positive rate (1-specificity) as the horizontal coordinate. The AUC value of the exosome marker combination IGF2+KRT20+SPAG5+NRP1 in the validation set is 0.902.

[0104] Figure 6. Screening results of differentially expressed genes.

[0105] Figure 7. Exosome morphology under transmission electron microscope.

[0106] Figure 8. NTA software quickly generates high-resolution particle size distribution of individual particles and the count of observed vesicle particles.

[0107] Figure 9. Amplification curve diagram of reaction combination 1 (IGF2+KRT20+SPAG5+NRP1) detecting bladder cancer positive samples.

[0108] Figure 10. Amplification curve diagram of reaction combination 2 (CDC20+DHRS2+ABL1) detecting bladder cancer positive samples.

[0109] Figure 11. Amplification curve diagram of reaction combination 3 (IGFBP5+VEGFA+MDK+CCNB1) detecting bladder cancer positive samples.

[0110] Figure 12. Amplification curve diagram of reaction combination 1 (IGF2+KRT20+SPAG5+NRP1) detecting bladder cancer negative samples.

[0111] Figure 13. Amplification curve diagram of reaction combination 2 (CDC20+DHRS2+ABL1) detecting bladder cancer negative samples.

[0112] Figure 14. Amplification curve diagram of reaction combination 3 (IGFBP5+VEGFA+MDK+CCNB1) detecting bladder cancer negative samples. DETAILED DESCRIPTION

[0113] The present application will be further described by way of examples, but the present application is not limited to the examples described. The experimental methods in the following examples are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0114] The urine exosome marker combination and kit for early detection of bladder cancer provided by the present application have the following steps in the specific implementation manner:

[0115] (1) Obtain urine samples from different subject groups. The different subject groups include patients diagnosed with bladder cancer, and patients without bladder cancer. The urine samples are the first urine excreted from the bladder, also known as "first-voided urine", with a collection volume of 30-50 mL.

[0116] (2) Extraction and characterization of exosomes in urine samples. In particular, exosomes are extracted using the company's patented product, EXODUS exosome purification system (Patent Publication No. CN114616054A). The resulting exosomes have the characteristics of high purity, high yield, and complete morphology.

[0117] For those skilled in the art, the extraction method of exosomes is not limited to the above operation steps. Suitable methods in the prior art such as ultracentrifugation, gradient density centrifugation, ultrafiltration centrifugation, magnetic bead immunization, etc., as well as the use of other commercial exosome precipitants are feasible. Those skilled in the art can predict that the resulting exosomes should have similar characteristics and there should be no difference due to changes in extraction methods.

[0118] In order to detect the characteristics of exosomes in the urine of bladder cancer patients and non-bladder cancer patients, transmission electron microscopy (Transmission electron microscope, TEM) was used to analyze the morphological characteristics of the extracted exosomes. The results are shown in Figure 7. The size distribution and particle concentration of nanometer particles in liquid suspension were obtained using light scattering and Brownian motion characteristics. The test results are shown in Figure 8.

[0119] (3) Exosome RNA extraction

[0120] The present application uses magnetic bead method nucleic acid extraction reagent to extract total nucleic acid from exosomes. The quality of the extracted RNA is controlled. The quality of the extracted nucleic acid can be evaluated by comparing the absorbance ratio (A260 / A280) of the nucleic acid sample at 260 nm and 280 nm. A260 / A280 is preferably 1.8-2.2, more preferably 2.0.

[0121] (4) Detection of nucleic acid biomarkers

[0122] The present application uses real-time fluorescent quantitative PCR method to measure the expression level of biomarkers in the above extracted RNA. The biomarkers include at least IGF2, KRT20, DHRS2 genes, and SPAG5 and / or NRP1 genes.

[0123] (5) Analysis of biomarker mRNA expression level

[0124] In the methods provided herein, the expression level of biomarker mRNA is determined by real-time fluorescent quantitative PCR analysis, and the positive reaction is detected by the accumulation of fluorescent signal. The Ct value is defined as the number of cycles required for the fluorescent signal to exceed the threshold value. The Ct value is inversely proportional to the amount of nucleic acid in the sample, i.e. the smaller the Ct value, the greater the amount of nucleic acid in the sample.

[0125] In the methods provided herein, the expression level of the detected gene is used to calculate the relative expression level of the gene, which is collectively referred to as the reference gene, for normalizing the signal value of the detected gene to control the differences between the amount of extracted exosomes, the performance of reagent components, and the performance of the fluorescent quantitative PCR instrument between samples. The reference gene is usually present in urine exosomes, and the present application is used to normalize the markers CCNB1, CDC20, SPAG5, NRP1, IGFBP5, IGF2, KRT20, VEGFA, MDK, and the reference genes ABL1 and DHRS2. The relative expression level analysis or normalization is completed by subtracting the Ct value of the reference gene (ABL1 or DHRS2) from the Ct value of CCNB1, CDC20, SPAG5, NRP1, IGFBP5, IGF2, KRT20, VEGFA, MDK, respectively, and the result is called ΔCt, for example:

[0126] ΔCt(IGF2) = Ct(IGF2) - Ct(DHRS2) or ΔCt(IGF2) = Ct(IGF2) - Ct(ABL1)

[0127] (6) Mining of biomarkers

[0128] The urine samples obtained from different subject populations are divided into two groups, i.e. cancer group and non-cancer group, and based on the relative expression level of each marker in the cancer group and the non-cancer group, statistical methods are used to mine mRNA biomarkers that can be used to distinguish bladder cancer and non-bladder cancer. The process is as follows: using the t-test method in the GraphPad Prism software to analyze the markers with significant differences in relative expression level between the cancer group and the non-cancer group, as candidate markers for subsequent analysis.

[0129] (7) Construction of bladder cancer prediction model

[0130] Receiver operating characteristic (ROC) curve is a widely used tool to evaluate the discrimination and diagnostic ability of biomarkers. Area under the curve (AUC) is established to evaluate the diagnostic value of each biomarker or marker combination. The biomarker or marker combination with the highest diagnostic value has an AUC value greater than 0.6, 0.7 or 0.8. Preferably, the individual marker or marker combination performance has an AUC value greater than 0.85. As shown in Figure 1, when IGF2, KRT20, SPAG5, NRP1 combination is determined together, the AUC of the model created by logistic regression analysis is 0.923 when the internal reference gene is DHRS2, and the multi-target combination has the highest diagnostic accuracy.

[0131] Further, the normalized expression levels of the above-mentioned IGF2, KRT20, SPAG5, NRP1 combination with higher diagnostic value are established by data analysis to establish a mathematical expression, and by combining the necessary sensitivity and specificity of clinical application, an early diagnosis model of bladder cancer is constructed. The algorithm of the model is:

[0132] Output value = {Ct(IGF2)-Ct(DHRS2)} x a + {Ct(KRT20)-Ct(DHRS2)} x b + {Ct(NRP1)+Ct(DHRS2)} x c + {Ct(SPAG5)+Ct(DHRS2)} x d + z.

[0133] In the formula, a, b, c, and d are coefficients, and z is a constant, which can be determined by fitting the output value of the equation to the existing data set by logistic regression or linear regression. The coefficient a is between -1 and 1, preferably the coefficient a = -0.468; the coefficient b is between -1 and 1, preferably the coefficient b = -0.473; the coefficient c is between -1 and 1, preferably the coefficient c = 0.318; the coefficient d is between -1 and 1, preferably the coefficient d = -0.280; and the constant z = 3.253.

[0134] The cutoff value of the output value determined by the ROC curve is used to distinguish the risk of bladder cancer in the subject. A value higher than the cutoff value indicates a higher risk of bladder cancer, and a value lower than the cutoff value indicates a lower risk of bladder cancer. The bladder cancer risk obtained by this method helps doctors make decisions on the next diagnostic options for patients, and serves as a supplement and aid to existing diagnostic methods for reference by clinicians.

[0135] Example 1 Bladder cancer detection kit amplifies urine exosome biomarkers

[0136] The target combination screening of bladder cancer detection kit exosome tumor markers is carried out, and a PCR reaction solution for RT-qPCR amplification is included, and the PCR reaction solution includes a set of primer and probe combinations for detecting 11 markers of bladder cancer urine exosomes, and the primer and probe combinations include: the primer pair (SEQ ID NO: 1, SEQ ID NO: 2) for amplifying the IGF2 gene at a concentration of 0.4 μM, and the probe (SEQ ID NO: 3) for amplifying the IGF2 gene at a concentration of 0.2 μM; the primer pair (SEQ ID NO: 4, SEQ ID NO: 5) for amplifying the KRT20 gene at a concentration of 0.4 μM, and the probe (SEQ ID NO: 6) for amplifying the KRT20 gene at a concentration of 0.1 μM; the primer pair (SEQ ID NO: 7, SEQ ID NO: 8) for amplifying the SPAG5 gene at a concentration of 0.4 μM, and the probe (SEQ ID NO: 9) for amplifying the SPAG5 gene at a concentration of 0.1 μM; the primer pair (SEQ ID NO: 10, SEQ ID NO: 11) for amplifying the NRP1 gene at a concentration of 0.4 μM, and the probe (SEQ ID NO: 12) for amplifying the NRP1 gene at a concentration of 0.2 μM; the primer pair (SEQ ID NO: 13, SEQ ID NO: 14) for amplifying the CDC20 gene at a concentration of 0.4 μM, and the probe (SEQ ID NO: 15) for amplifying the CDC20 gene at a concentration of 0.1 μM; the primer pair (SEQ ID NO: 16, SEQ ID NO: 17) for amplifying the CCNB1 gene at a concentration of 0.4 μM, and the probe (SEQ ID NO: 18) for amplifying the CCNB1 gene at a concentration of 0.1 μM; the primer pair (SEQ ID NO: 19, SEQ ID NO: 20) for amplifying the IGFBP5 gene at a concentration of 0.4 μM, and the probe (SEQ ID NO: 21) for amplifying the IGFBP5 gene at a concentration of 0.2 μM; the primer pair (SEQ ID NO: 22, SEQ ID NO: 23) for amplifying the VEGFA gene at a concentration of 0.4 μM, and the probe (SEQ ID NO: 24) for amplifying the VEGFA gene at a concentration of 0.1 μM; the primer pair (SEQ ID NO: 25, SEQ ID NO: 26) for amplifying the MDK gene at a concentration of 0.4 μM, and the probe (SEQ ID NO: 27) for amplifying the MDK gene at a concentration of 0.2 μM; the primer pair (SEQ ID NO: 28, SEQ ID NO: 29) for amplifying the DHRS2 gene at a concentration of 0.4 μM, and the primer pair (SEQ ID NO: 30) for amplifying the DHRS2 gene at a concentration of 0.1 μM; the primer pair (SEQ ID NO: 31, SEQ ID NO: 32) for amplifying the ABL1 gene at a concentration of 0.4 μM, and the probe (SEQ ID NO: 33) for amplifying the ABL1 gene at a concentration of 0.2 μM of the probe (SEQ ID NO: 33) for amplifying ABL1 gene.

[0137] In this embodiment, the fluorescent group of the probe shown in SEQ ID NO: 3 is HEX, and the quenching group is BHQ1; the fluorescent group of the probe shown in SEQ ID NO: 6 is ROX, and the quenching group is BHQ2; the fluorescent group of the probe shown in SEQ ID NO: 9 is CY5, and the quenching group is BHQ2; the fluorescent group of the probe shown in SEQ ID NO: 12 is FAM, and the quenching group is BHQ1; the fluorescent group of the probe shown in SEQ ID NO: 15 is HEX, and the quenching group is BHQ1; the fluorescent group of the probe shown in SEQ ID NO: 18 is FAM, and the quenching group is BHQ1; the fluorescent group of the probe shown in SEQ ID NO: 21 is HEX, and the quenching group is BHQ1; the fluorescent group of the probe shown in SEQ ID NO: 24 is CY5, and the quenching group is BHQ2; the fluorescent group of the probe shown in SEQ ID NO: 27 is ROX, and the quenching group is BHQ2; the fluorescent group of the probe shown in SEQ ID NO: 30 is FAM, and the quenching group is BHQ1; the fluorescent group of the probe shown in SEQ ID NO: 33 is ROX, and the quenching group is BHQ2; see Table 1 for the partial PCR amplification system.

[0138] Table 1 PCR amplification reaction system

[0139] The PCR reaction solution is composed of the mixture of the above-mentioned primer and probe combination, and the PCR buffer solution containing the necessary magnesium ions, dA / G / C / UTPs for the PCR reaction. In addition to the above, the kit also includes an enzyme mixture composed of reverse transcriptase, DNA polymerase and UNG enzyme.

[0140] When preparing the PCR amplification reaction solution, the formula is as follows: 18.5 microliters of the PCR reaction solution containing the above-mentioned primer and probe combination per test, 1.5 microliters of the enzyme mixture per test, 10 microliters of the sample to be tested per test, and the total volume is 30 microliters.

[0141] After the reagents are prepared, they are placed in an ABI 7500 fluorescent quantitative PCR instrument for reaction, and the PCR reaction conditions are set according to Table 2.

[0142] Table 2 PCR amplification reaction conditions

[0143] After the PCR reaction, the Ct value of each RNA target was analyzed using the ABI 7500 software. FIG. 9, FIG. 10, and FIG. 11 are amplification curve diagrams of reaction combination 1, reaction combination 2, and reaction combination 3 on bladder cancer positive samples, respectively, and FIG. 12, FIG. 13, and FIG. 14 are amplification curve diagrams of reaction combination 1, reaction combination 2, and reaction combination 3 on bladder cancer negative samples.

[0144] Example 2 Screening of a urine exosome marker combination for bladder cancer detection and model construction

[0145] The present embodiment provides a method for screening and constructing a prediction model of a urine exosome marker combination for bladder cancer detection, and statistical verification of patient samples.

[0146] In this study, samples were used if the following criteria were met:

[0147] The inclusion criteria were as follows: 1) age between 18 and 80 years old, gender not limited; 2) signed informed consent for clinical trial; 3) bladder cancer group: intended to be diagnosed as bladder cancer by at least one pathological means within 2 weeks in the center; 4) normal group: control group of patients without bladder malignancies, including bladder inflammation, etc., 5) agreed to leave the morning urine or urine with an interval of 2-3 hours for research; any outpatient or inpatient volunteer who met all the above criteria could be included as a subject;

[0148] The exclusion criteria were as follows: 1) age less than 18 years old or more than 80 years old; unwilling to sign the informed consent; 2) patients with a history of bladder malignancy; 3) patients who could not understand the research process, or could not / would not answer the questionnaire, provide medical records, examination reports, and other relevant information; 4) patients with a history of AIDS infection; 5) patients with other malignancies (within 3 years); 6) patients taking antitumor drugs (within 3 months); 7) patients with severe hematuria and infection that would affect the test results according to the judgment of the researchers; any patient who met any of the above criteria could not be included as a subject.

[0149] According to the above criteria, a total of 453 patients were recruited for the study as training set samples, and the sample statistical information was as follows:

[0150] The screening and prediction model construction method of the above biomarker combination is as follows:

[0151] (1) The above 453 samples were subjected to exosome enrichment and purification using the method described in the patent application (CN202280000660). Exosome biomarker extraction and RT-qPCR detection were performed using techniques well known in the art to obtain the detection Ct value of the exosome biomarker. The biomarker at least comprises IGF2, KRT20, SPAG5, NRP1, and DHRS2.

[0152] (2) Biomarker mRNA expression level analysis, gene expression level normalization.

[0153] (3) Screening of differentially expressed genes. The normalized expression differences of IGF2, KRT20, SPAG5, NRP1, etc. in 225 non-bladder cancer samples and 228 bladder cancer samples were analyzed by t-test method. As shown in Figure 6, the target CCNB1, CDC20, SPAG5, NRP1, IGFBP5, IGF2, KRT20, MDK, VEGTA showed significant differences in cancer population and non-cancer population (including hematuria patients), which had certain diagnostic value for bladder cancer diagnosis.

[0154] (4) According to the significantly different expression genes, the diagnostic performance of different markers or marker combination models was evaluated by logistic regression, and the results were as shown in Table 3:

[0155] Table 3 Performance of different markers or marker combinations

[0156] In the logistic regression analysis, the four targets IGF2+KRT20+SPAG5+NRP1 and the internal reference DHRS2 prediction model, as shown in Figures 1-2, the maximum AUC was 0.923, and the sensitivity (Sensitivity) and specificity (Specificity) for diagnosing bladder cancer were 85.5% and 84.4%, respectively.

[0157] The urine sample is obtained by a non-invasive method, and the cell precipitate does not need to be separated from the urine sample. By combined detection of bladder cancer related gene expression detection, the early tumor detection rate can be significantly improved, and the bladder cancer exosome detection has the characteristics of non-invasive, accurate and rapid.

[0158] Example 3 Application of bladder cancer prediction model in validation set

[0159] This embodiment provides a use method of a bladder cancer early screening model based on urine exosomes, which can also be used to verify the accuracy of the bladder cancer prediction model based on urine provided in Example 2. The results are shown in Figures 3-4.

[0160] The above kit, method and logistic regression formula were selected to perform exosome nucleic acid detection analysis on a total of 191 urine samples of suspected bladder cancer from outpatients of the Department of Urology of Wuhan Tongji Hospital and the First Affiliated People's Hospital of Wenzhou.

[0161] According to the hospital clinical biopsy and pathological diagnosis results, 91 cases of bladder cancer urine samples and 100 cases of non-bladder cancer urine samples were obtained. According to the obtained logistic regression calculation formula, the comprehensive score was calculated by using the set of proprietary algorithms to evaluate the risk of 191 patients suffering from bladder cancer. The detection results of the validation set are shown in Tables 4 and 5.

[0162] Table 4 Performance of different markers or marker combinations in the validation set

[0163] Table 5 Diagnostic performance of target combination IGF2+KRT20+SPAG5+NRP1 compared with pathological results

[0164] Sensitivity = 79 / 91 = 86.8%;

[0165] Specificity = 83 / 100 = 83.0%;

[0166] Overall agreement = (79+83) / 191 = 84.8%;

[0167] As shown in Figure 5, the AUC of the exosome marker combination IGF2+KRT20+SPAG5+NRP1 is 0.902, and the exosome marker combination has better diagnostic performance. Among the 91 cases of bladder cancer urine samples, the proportion of high-grade cancer patients included is relatively small, and the AUC of the validation set is lower than that of the training set, which is 0.923.

[0168] The accuracy of the bladder cancer early screening model is verified by using clinical samples, and the results show that the data basically conforms to the previous data, which can meet the requirements of clinical detection, and can increase the detection rate of early cancer detection for patients.

[0169] Exosome samples are easy and fast to extract, have minimal risk and low cost, and can reduce the pain of patients; exosomes can be used to monitor cancer treatment results or monitor cancer recurrence, increase the detection rate of bladder cancer in high-risk populations, and find early bladder cancer; reduce the mortality rate of bladder cancer in the screening population without affecting the quality of life of the screening population.

[0170] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent substitutions or changes to the technical solutions and inventive concepts of the present application within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. Use of a reagent for detecting a biomarker in the manufacture of a test product, characterized in that, The detection product is used for detecting bladder cancer; the biomarker comprises a cell proliferation regulatory factor and a keratin.

2. Use according to claim 1, characterized in that, The cell proliferation regulatory factor is selected from IGF2, and the keratin is selected from KRT20.

3. Use according to claim 2, characterized in that, The biomarker further comprises one or more genes selected from SPAG5, NRP1, CCNB1, CDC20, IGFBP5, VEGFA and MDK.

4. Use according to claim 2 or 3, characterized in that, The biomarker consists of IGF2, KRT20, SPAG5 and NRP1.

5. The use according to claim 1, characterized in that, The biomarker further comprises a reference gene selected from DHRS2 or ABL1.

6. A primer set, characterized in that, The primer set is used for detecting a biomarker comprising a cell proliferation regulatory factor and a keratin; the primer set comprises: primers for amplifying IGF2, wherein the upstream primer sequence is shown as SEQ ID NO: 1, and the downstream primer sequence is shown as SEQ ID NO: 2; primers for amplifying KRT20, wherein the upstream primer sequence is shown as SEQ ID NO: 4, and the downstream primer sequence is shown as SEQ ID NO: 5; primers for amplifying SPAG5, wherein the upstream primer sequence is shown as SEQ ID NO: 7, and the downstream primer sequence is shown as SEQ ID NO: 8; primers for amplifying NRP1, wherein the upstream primer sequence is shown as SEQ ID NO: 10, and the downstream primer sequence is shown as SEQ ID NO: 11; primers for amplifying CDC20, wherein the upstream primer sequence is shown as SEQ ID NO: 13, and the downstream primer sequence is shown as SEQ ID NO: 14; primers for amplifying CCNB1, wherein the upstream primer sequence is shown as SEQ ID NO: 16, and the downstream primer sequence is shown as SEQ ID NO: 17; primers for amplifying IGFBP5, wherein the upstream primer sequence is shown as SEQ ID NO: 19, and the downstream primer sequence is shown as SEQ ID NO: 20; primers for amplifying VEGFA, wherein the upstream primer sequence is shown as SEQ ID NO: 22, and the downstream primer sequence is shown as SEQ ID NO: 23; primers for amplifying MDK, wherein the upstream primer sequence is shown as SEQ ID NO: 25, and the downstream primer sequence is shown as SEQ ID NO:

26.

7. The primer set according to claim 6, wherein The primer set further comprises: primers for amplifying the reference gene DHRS2, wherein the upstream primer sequence is shown as SEQ ID NO: 28, and the downstream primer sequence is shown as SEQ ID NO: 29; primers for amplifying the reference gene ABL1, wherein the upstream primer sequence is shown as SEQ ID NO: 31, and the downstream primer sequence is shown as SEQ ID NO:

32.

8. A probe set, characterized in that, The probe set is used for detecting a biomarker comprising a cell proliferation regulatory factor and a keratin; the probe set comprises: The probe sequence for detecting IGF2 is shown as SEQ ID NO: 3, the probe sequence for detecting KRT20 is shown as SEQ ID NO: 6, the probe sequence for detecting SPAG5 is shown as SEQ ID NO: 9, the probe sequence for detecting NRP1 is shown as SEQ ID NO: 12, the probe sequence for detecting CDC20 is shown as SEQ ID NO: 15, the probe sequence for detecting CCNB1 is shown as SEQ ID NO: 18, the probe sequence for detecting IGFBP5 is shown as SEQ ID NO: 21, the probe sequence for detecting VEGFA is shown as SEQ ID NO: 24, the probe sequence for detecting MDK is shown as SEQ ID NO: 27, the probe sequence for detecting DHRS2 is shown as SEQ ID NO: 30, and the probe sequence for detecting ABL1 is shown as SEQ ID NO:

33.

9. The probe set of claim 8, wherein, The probes of the probe set are labeled with fluorescent reporter groups selected from FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3 or JOE.

10. The probe set of claim 9, wherein The fluorescent reporter group of the probe shown as SEQ ID NO: 3 is HEX; the fluorescent reporter group of the probe shown as SEQ ID NO: 6 is ROX; the fluorescent reporter group of the probe shown as SEQ ID NO: 9 is CY5; and the fluorescent reporter group of the probe shown as SEQ ID NO: 12 is FAM.

11. The probe set of claim 8, wherein The 3' end of the probes of the probe set also has a fluorescent quenching group selected from BHQ1 or BHQ2.

12. The probe set according to claim 11, wherein, The fluorescent quenching group of the probe shown as SEQ ID NO: 3 is BHQ1; the fluorescent quenching group of the probes shown as SEQ ID NO: 6 and SEQ ID NO: 9 is BHQ2; and the fluorescent quenching group of the probe shown as SEQ ID NO: 12 is BHQ1.

13. The probe set according to claim 11, wherein, The fluorescent reporter group of the probe shown as SEQ ID NO: 15 is HEX, and the fluorescent quenching group is BHQ1; the fluorescent reporter group of the probe shown as SEQ ID NO: 18 is FAM, and the fluorescent quenching group is BHQ1; the fluorescent reporter group of the probe shown as SEQ ID NO: 21 is HEX, and the fluorescent quenching group is BHQ1; the fluorescent reporter group of the probe shown as SEQ ID NO: 24 is CY5, and the fluorescent quenching group is BHQ2; the fluorescent reporter group of the probe shown as SEQ ID NO: 27 is ROX, and the fluorescent quenching group is BHQ2; the fluorescent reporter group of the probe shown as SEQ ID NO: 30 is FAM, and the fluorescent quenching group is BHQ1; and the fluorescent reporter group of the probe shown as SEQ ID NO: 33 is ROX, and the fluorescent quenching group is BHQ2.

14. A kit characterized in that, The kit is used for detecting biomarkers including cell proliferation regulatory factors and keratins; the kit comprises the primer set of claim 6 or 7 and the probe set of any one of claims 8-13.

15. A kit according to claim 14, wherein The kit comprises reagents for detecting urine exosomes and extracting urine exosomes.

16. The kit of claim 15, wherein The kit comprises: The RT-qPCR reaction solution comprises primers and probes specifically recognizing mRNA sequences of IGF2, KRT20, SPAG5, NRP1 and DHRS2, an enzyme mixture comprising reverse transcriptase, DNA polymerase and UDG enzyme, positive quality control and negative quality control.

17. A kit according to claim 16, wherein The final concentration of the primers in the RT-qPCR reaction system is 0.2-0.4 μM, and the final concentration of the probes in the RT-qPCR reaction system is 0.1-0.3 μM.

18. A kit according to claim 17, wherein The RT-qPCR reaction system further comprises Tris buffer, magnesium ions, dA / G / C / UTPs; the final concentration of the magnesium ions is 5 mM, the final concentration of the dATP is 0.4 mM, the final concentration of the dGTP is 0.4 mM, the final concentration of the dCTP is 0.4 mM, and the final concentration of the dUTP is 0.8 mM.

19. A non-diagnostic method of bladder cancer based on a combination of urinary exosomal biomarkers, characterized in that, The method is used for converting the marker combination gene expression level into the bladder cancer risk level of the patient to be tested, and the model algorithm is: Output value = {Ct(Target 1)-Ct(internal reference)}×a+{Ct(Target 2)-Ct(internal reference)}×b+{Ct(Target 3)-Ct(internal reference)}×c+{Ct(Target 4)-Ct(internal reference)}×d+…+{Ct(Target N)-Ct(internal reference)}×n+z; In the formula, a, b, c, …, n are coefficients, z is a constant, the coefficients are between -1 and 1, Ct(internal reference) is the Ct value of the internal reference gene, and Ct(Target 1) to Ct(Target N) are the Ct values of the genes in the marker combination of claims 1-5.

20. The method of claim 19, wherein, The kit of any one of claims 14-18 is used for detecting the mRNA expression levels of IGF2, KRT20, SPAG5, NRP1 and DHRS2 in urine exosomes, then the model is used to calculate the output value and compare it with the positive judgment value, and the risk level of the subject suffering from bladder cancer is analyzed.

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