Nucleic acid combination product for detecting bladder cancer or urothelial cancer and use thereof

Abstract

The application relates to the biomedical technology field, in particular to a nucleic acid combination product for detecting bladder cancer or urothelial cancer and application thereof. The nucleic acid combination product of the application adds a primer pair and a probe for detecting at least one of two auxiliary genes NID2 and PENK on the basis of a primer pair and a probe for detecting a single TWIST1 gene, improves the positive detection rate and accuracy of bladder cancer and urothelial cancer, and provides more possibilities for early detection of tumor diseases and treatment thereof.

Description

[0001] This application claims priority to the Chinese patent application No. 2024103225640, filed on March 20, 2024, and entitled “Nucleic acid combination product for detecting bladder cancer and application thereof”, the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of biomedical technology, in particular to a nucleic acid combination product for detecting bladder cancer or urothelial carcinoma and application thereof. BACKGROUND

[0003] Bladder cancer (BC) is the 10th most common cancer worldwide, with approximately 570,000 new cases and more than 210,000 deaths each year. The routine clinical examinations for bladder cancer include urine routine, B-ultrasound, urine exfoliative cytology, and cystoscopy FISH, etc. Cystoscopy may cause risks such as urinary tract infection, urethral injury, and bladder injury. Ordinary cystoscopy has poor patient compliance, while painless cystoscopy has the risk of anesthesia, higher charges, and cannot avoid the risk of urinary tract infection. The sensitivity or specificity of B-ultrasound and urine exfoliative cytology is not high, and they cannot accurately identify patients, especially those in the early stage of cancer.

[0004] Traditional early screening methods have many limitations. If we want to effectively improve the early diagnosis rate of bladder cancer, new technologies are needed to break the ice. Liquid biopsy is a cancer early diagnosis technology that has been highly anticipated in recent years. It is a gene detection method that uses non-invasive methods to collect DNA from body fluids to diagnose tumors. The main biomarkers include circulating tumor cells (CTC), ctDNA, and exosomes (Exosome). For example, by detecting the gene methylation status of tumor cells exfoliated in urine to assist in evaluating the occurrence, recurrence, and drug efficacy of bladder cancer.

[0005] A variety of factors are associated with the recurrence and progression of bladder cancer and affect patient survival. There are currently a variety of molecular markers for checking bladder cancer, but their value is very limited. Therefore, new molecular markers are needed to detect bladder cancer patients, especially those at high risk of progression and recurrence. Recent epigenetic markers can be used as an assessment of high risk of progression to cancer, providing new opportunities for treatment and prevention. DNA methylation as a biomarker has received increasing attention in recent years because abnormal DNA methylation is a major feature of bladder cancer and plays a key role in the occurrence and progression of bladder cancer.

[0006] Genetics is the study of hereditary information based on genes, while epigenetics is the study of reversible changes in gene function that can be inherited, without any changes in the DNA itself. DNA methylation occurs throughout the genome, including the addition of a methyl group to the cytosine ring of CpG dinucleotides by methyltransferases. DNA methylation is a key regulator of gene transcription and genome stability. In human cancers, abnormal changes in DNA methylation patterns, activation of oncogenes with high expression, and silencing of tumor suppressor genes with no expression, cause an imbalance between oncogenes and tumor suppressors, ultimately leading to cancer. Many cancers show global hypomethylation, and some genes (proven to be tumor suppressors) have highly methylated regions in their promoters, making it possible to use methylation as a marker for cancer. In addition, each tumor type may have its own unique methylation pattern, which can be used to develop various tumor-specific methylation markers. Changes in epigenetic events occur during the carcinogenic stage or early stages of cancer, so genetic methylation as a molecular diagnosis may help detect cancer before symptoms or obvious imaging manifestations appear.

[0007] Great progress has been made in the study of epigenetics of bladder cancer. Since high methylation of tumor suppressor gene promoters is common in bladder cancer, potential DNA methylation markers have been identified in serum, bladder irrigation fluid, urine samples, and cancer tissues. Therefore, DNA methylation can be used as a biomarker for early detection, effective treatment, and accurate prognosis of bladder cancer.

[0008] A single-center study used MS-qPCR to detect 4 methylation sites (VIM, RASSF1A, GDF15, and TMEFF2) to diagnose bladder cancer, with a sensitivity of 82% and a specificity of 53%. Previous studies have shown that DNA methylation in urine is an effective marker for the diagnosis of bladder cancer, but its sensitivity and specificity still need to be improved.

[0009] Recently, the team of Huang Jian / Lin Tianqi published a research report entitled "Urine DNA methylation assay enables early detection and recurrence monitoring for bladder cancer" in the Journal of Clinical Investigation, written by Chen Xu. This study integrated and analyzed sequencing data from 3 cohorts of bladder cancer, identified 26 bladder cancer-specific methylation sites, and then used a single-center cohort of 313 cases and a prospective multicenter cohort of 175 cases to model and verify 2 methylation sites. The AUC of the training group and the validation group was 0.919 and 0.903, respectively, with an overall accuracy of 86.7% for bladder cancer patients, a sensitivity of 90.0%, and a specificity of 83.1%.

[0010] Overall, the current non-invasive urinary biomarker detection method for bladder cancer is not ideal in terms of sensitivity and specificity, with high rates of missed diagnosis and misdiagnosis, making it difficult to meet clinical needs and resulting in a low rate of clinical practical application.

[0011] Urothelial carcinoma (UC) is a relatively common malignant tumor worldwide. Early diagnosis is crucial for the prognosis of urothelial carcinoma patients, as the five-year survival rate of patients with localized urothelial carcinoma can exceed 90%. However, once muscle layer invasion or metastasis occurs, the survival period and quality of life of patients will significantly decrease.

[0012] Currently, the diagnostic methods for urothelial carcinoma include cystoscopy, urine exfoliative cytology, fluorescence in situ hybridization (FISH), etc. However, these methods have certain limitations. For example, although cystoscopy combined with biopsy is the "gold standard" for the diagnosis of urothelial carcinoma, it is not suitable for large-scale screening due to its high invasiveness and high cost. Therefore, researchers are seeking more convenient, non-invasive, sensitive, and specific early diagnostic methods.

[0013] In recent years, urine DNA detection has shown great potential as a non-invasive detection method. By detecting DNA copy number variation and methylation variation in urine, tumors and normal tissues can be accurately predicted. Professor He Zhisong's team from Peking University First Hospital, Professor Ye Dingwei's team from Fudan University Cancer Hospital, etc. cooperated with Jinjia Medicine to publish the data of the world's first urine urothelial carcinoma early screening and efficacy monitoring product, meeting the needs of more accurate, more convenient, and non-invasive urothelial carcinoma diagnosis and treatment. In addition, the Urothelial Carcinoma Expert Committee of the Chinese Society of Clinical Oncology (CSCO) released the "Urothelial Carcinoma Diagnosis and Treatment Guidelines (2020 Edition)", which provides an important reference for the domestic urothelial carcinoma diagnosis and treatment field, including guidance for early diagnosis.

[0014] Overall, the research background of early diagnosis of urothelial carcinoma focuses on improving the accuracy of diagnosis, reducing invasiveness, and increasing patient compliance, while emphasizing the combination of domestic and foreign research results and Chinese clinical practice in order to improve the prognosis and quality of life of patients.

[0015] In view of the above, the present application is proposed. SUMMARY

[0016] One or more embodiments of the present application provide a nucleic acid combination product for detecting bladder cancer or urothelial carcinoma and its application, which can be used for the diagnosis of bladder cancer and urothelial carcinoma, has good specificity and high sensitivity.

[0017] One or more embodiments of the present application provide a nucleic acid combination product comprising a primer pair and a probe for detecting a biomarker;

[0018] The primer pair and the probe for detecting the biomarker comprise a primer pair and a probe for detecting a TWIST1 gene, and further comprise a primer pair and a probe for detecting one or more of a PENK gene and a NID2 gene;

[0019] The primer pair and the probe for detecting the TWIST1 gene comprise a forward primer represented by any one of SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 10 and SEQ ID NO. 13, a reverse primer represented by any one of SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 8, SEQ ID NO. 11 and SEQ ID NO. 14, and a probe represented by any one of SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12 and SEQ ID NO. 15;

[0020] The primer pair and the probe for detecting the PENK gene comprise a forward primer represented by any one of SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22, SEQ ID NO. 25 and SEQ ID NO. 28, a reverse primer represented by any one of SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23, SEQ ID NO. 26 and SEQ ID NO. 29, and a probe represented by any one of SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 27 and SEQ ID NO. 30;

[0021] The primer pair and the probe for detecting the NID2 gene comprise a forward primer represented by any one of SEQ ID NO. 31 and SEQ ID NO. 34, a reverse primer represented by any one of SEQ ID NO. 32 and SEQ ID NO. 35, and a probe represented by any one of SEQ ID NO. 33 and SEQ ID NO. 36.

[0022] In some embodiments of the present application, the primer pair and the probe for detecting the TWIST1 gene comprise:

[0023] a forward primer represented by SEQ ID NO. 1, a reverse primer represented by SEQ ID NO. 2, and a probe represented by SEQ ID NO. 3; or,

[0024] a forward primer of SEQ ID NO. 4, a reverse primer of SEQ ID NO. 5, and a probe of SEQ ID NO. 6; or,

[0025] a forward primer of SEQ ID NO. 7, a reverse primer of SEQ ID NO. 8, and a probe of SEQ ID NO. 9; or,

[0026] a forward primer of SEQ ID NO. 10, a reverse primer of SEQ ID NO. 11, and a probe of SEQ ID NO. 12; or,

[0027] a forward primer of SEQ ID NO. 13, a reverse primer of SEQ ID NO. 14, and a probe of SEQ ID NO. 15.

[0028] In some embodiments of the present application, the primer pair and the probe for detecting the PENK gene comprise:

[0029] a forward primer of SEQ ID NO. 16, a reverse primer of SEQ ID NO. 17, and a probe of SEQ ID NO. 18; or,

[0030] a forward primer of SEQ ID NO. 19, a reverse primer of SEQ ID NO. 20, and a probe of SEQ ID NO. 21; or,

[0031] a forward primer of SEQ ID NO. 22, a reverse primer of SEQ ID NO. 23, and a probe of SEQ ID NO. 24; or,

[0032] a forward primer of SEQ ID NO. 25, a reverse primer of SEQ ID NO. 26, and a probe of SEQ ID NO. 27; or,

[0033] a forward primer of SEQ ID NO. 28, a reverse primer of SEQ ID NO. 29, and a probe of SEQ ID NO. 30.

[0034] In some embodiments of the present application, the primer pair and the probe for detecting the NID2 gene comprise:

[0035] a forward primer of SEQ ID NO. 31, a reverse primer of SEQ ID NO. 32, and a probe of SEQ ID NO. 33; or,

[0036] a forward primer represented by SEQ ID NO. 34, a reverse primer represented by SEQ ID NO. 35, and a probe represented by SEQ ID NO. 36.

[0037] In some embodiments of the present application, the primer pair and the probe for detecting the biomarker consist of the primer pair and the probe for detecting the TWIST1 gene and the primer pair and the probe for detecting the PENK gene, or consist of the primer pair and the probe for detecting the TWIST1 gene and the primer pair and the probe for detecting the NID2 gene.

[0038] In some embodiments of the present application, the primer pair and the probe for detecting the biomarker consist of the primer pair and the probe for detecting the TWIST1 gene and the primer pair and the probe for detecting the NID2 gene.

[0039] In some embodiments of the present application, the 5' end of the probe in the primer pair and the probe for detecting the biomarker is labeled with a fluorescent group, and the 3' end is labeled with a quenching group; optionally, the fluorescent groups of the probes for detecting different biomarkers have different excitation light waves.

[0040] In some embodiments of the present application, the nucleic acid combination product further comprises a primer pair and a probe for detecting a reference gene.

[0041] Optionally, the primer pair and the probe for detecting the reference gene comprise a forward primer represented by SEQ ID NO. 37, a reverse primer represented by SEQ ID NO. 38, and a probe represented by SEQ ID NO. 39.

[0042] Further optionally, the 5' end of the probe in the primer pair and the probe for detecting the reference gene is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

[0043] Still further, the probe in the primer pair and the probe for detecting the reference gene and the probe in the primer pair and the probe for detecting the biomarker have fluorescent groups with different excitation light waves.

[0044] Still another or more embodiments of the present application provide use of the nucleic acid combination product in the preparation of a bladder cancer diagnostic kit or a urothelial carcinoma diagnostic kit.

[0045] Still another or more embodiments of the present application provide a kit comprising the nucleic acid combination product.

[0046] In some embodiments of the present application, the kit further comprises one or more of a DNA amplification reagent, a reagent for converting unmethylated cytosine bases to uracil, a DNA extraction reagent, and a DNA purification reagent.

[0047] Details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the present application will be apparent from the description. BRIEF DESCRIPTION OF DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings needed to be used in the embodiments. Obviously, the drawings in the following description only some embodiments of the present application, and for those skilled in the art, on the premise of not paying the creative labor, can also obtain other drawings according to these drawings.

[0049] Figure 1 ROC curve for TWIST1 gene and PENK gene combined detection;

[0050] Figure 2 ROC curve for TWIST1 gene and NID2 gene combined detection;

[0051] Figure 3 Detection results corresponding to the nucleic acid combination 1;

[0052] Figure 4 Detection results corresponding to the nucleic acid combination 2;

[0053] Figure 5 Detection results corresponding to the nucleic acid combination 3;

[0054] Figure 6 Detection results corresponding to the nucleic acid combination 4;

[0055] Figure 7 Detection results corresponding to the nucleic acid combination 5;

[0056] Figure 8 Detection results corresponding to the nucleic acid combination 6;

[0057] Figure 9 Detection results corresponding to the nucleic acid combination 7;

[0058] Figure 10 Detection results corresponding to the nucleic acid combination 8;

[0059] Figure 11 Detection results corresponding to the nucleic acid combination 9;

[0060] Figure 12 Detection results corresponding to the nucleic acid combination 10;

[0061] Figure 13 Detection results corresponding to the nucleic acid combination 11;

[0062] Figure 14 Detection results corresponding to the nucleic acid combination 12;

[0063] Figure 15 Detection results of the nucleic acid combination 5 and the nucleic acid combination 10 combined detection;

[0064] Figure 16 Results for the combined detection of TWIST1 and NID2. DETAILED DESCRIPTION

[0065] The application will be described in further detail with reference to the drawings, embodiments and examples. It should be understood that these embodiments and examples are intended to illustrate the application and are not intended to limit the scope of the application. The purpose of these embodiments and examples is to make the disclosure of the application more thorough and complete. It should also be understood that the application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the application, and the equivalent forms obtained thereby also fall within the protection scope of the application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the application. It should be understood that the application can be implemented without one or more of these details.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing embodiments and examples only and is not intended to be limiting of the application.

[0067] Terminology

[0068] Unless otherwise indicated or unless contradicted by context, terms or phrases used herein have the following meanings:

[0069] The term "and / or", "or / and", "and / or" used herein is a selective range including any one of two or more related listed items, and also including any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in this application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0070] In the present application, "multiple", "various", "multiple times", "multiple", etc. refer to more than two or equal to two in number without specific limitation. For example, "one or more" means one or more than two.

[0071] As used herein, "combinations thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0072] As used herein, "suitable combinations", "suitable ways", "any suitable way" and the like mean that the technical solutions of the present application can be implemented, the technical problems of the present application can be solved, and the intended technical effects of the present application can be achieved.

[0073] As used herein, "preferably", "better", "better", "as appropriate" only describe the better effect of the embodiments or examples, and it should be understood that it does not constitute a limitation on the scope of protection of the present application.

[0074] In the present application, "further", "more further", "particularly" and the like are used to describe the purpose, indicating the difference in content, but should not be understood as a limitation on the scope of protection of the present application.

[0075] In the present application, "optionally", "optional", "optional" means optional, that is, selected from "yes" or "no" two parallel schemes. If there are multiple "optional" in a technical solution, unless otherwise specified, and there is no contradiction or mutual restriction, each "optional" is independent.

[0076] In the present application, "first aspect", "second aspect", "third aspect", "fourth aspect" and the like, the terms "first", "second", "third", "fourth" and the like are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on the quantity.

[0077] In the present application, the technical features described in an open manner include both the closed technical solutions consisting of the listed features and the open technical solutions containing the listed features.

[0078] In the present application, when a numerical interval (i.e. a numerical range) is involved, unless otherwise specified, the optional numerical distribution within the numerical interval is considered to be continuous, and includes both numerical end points (i.e. the minimum value and the maximum value) of the numerical range, and every numerical value between the two numerical end points. When a numerical interval refers to only integers within the numerical interval, unless otherwise specified, including both end point integers of the numerical range, and every integer between the two end point integers, in the present application, it is equivalent to directly listing every integer, for example, t is an integer selected from 1 to 10, which means that t is any one integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in the present application should be understood to include any and all sub-ranges incorporated therein.

[0079] In the present application, unless otherwise specified, the temperature parameter allows both constant temperature treatment and fluctuation within a certain temperature interval. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuation within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C is allowed.

[0080] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0081] All the documents mentioned in the present application are cited in the present application as references, as if each document is cited as a reference individually. Unless and to the extent that the cited documents conflict with the purpose and / or technical solution of the present application, the cited documents are cited in the present application in their entirety and for all purposes. When the present application refers to the cited documents, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited in the present application. When the present application refers to the cited documents, the examples and preferred modes of the relevant technical features cited are also incorporated into the present application as references, but are limited to the implementation of the present application. It should be understood that when the cited content conflicts with the description in the present application, the present application is given priority or is modified adaptively according to the description in the present application.

[0082] In the reported literature, the sensitivity of single gene methylation detection for bladder cancer is 65%-100%, and the same gene in different studies has a wide range of clinical sensitivity. The factors considered include ① differences in the population enrolled, which is affected by the disease spectrum of each research institution; ② the same gene is detected by each study, but the methylation sites in the promoter region are different; ③ differences in research types, retrospective and prospective studies, which bring bias to the test results (Kim and Kim 2016). The genes reported in the traditional detection of bladder cancer, such as TWIST1, DAPK1, TERT, BCL2, p16, RASSF1A, COL1A2, have poor detection effect.

[0083] Early detection of bladder cancer (BCa) provides favorable outcomes for patients and avoids the need for cystectomy. Development of accurate and sensitive non-invasive BCa diagnostic test agents is imperative. DNA methylation is an early epigenetic event in the development of BCa. Certain specific aberrant methylation can serve as useful biomarkers. The purpose of this application is to determine methylation biomarkers for early detection of BCa. CpG methylation microarray analysis was performed on primary tumors and paired non-tumor tissues from 9 BCa patients with different stages (T1-T4). Bisulfite pyrosequencing was performed to confirm the methylation status of candidate genes in tissues and urine sediments (n=51). Among them, PENK was selected as a potential candidate, and a set of independent 169 urine sediments (55 BCa, 25 benign urological diseases, 8 other urological cancers and 81 healthy controls) were used with quantitative methylation-specific real-time PCR (mePENK-qMSP). All statistical analyses were performed using MedCalc software version 9.3.2.0. CpG methylation microarray analysis and stepwise validation of tissues and urine sediments by bisulfite pyrosequencing support the aberrant methylation site of the PENK gene as a potential biomarker for early detection of BCa. Clinical validation of the mePENK-qMSP test using urine sediment DNA showed a sensitivity of 86.5% (95% CI: 71.2-95.5%) and a specificity of 92.5% (95% CI: 85.7-96.7%). The area under the ROC was 0.920 (95% CI: 0.863-0.959) in detecting Ta high-grade and advanced tumor stages (T1-T4) of BCa patients. The sensitivities of low-grade papillary, high-grade papillary, T1 and T2-T4 were 55.6, 83.3, 88.5 and 100%, respectively. The methylation status of PENK was not related to gender, age or stage, but was related to the tumor grade of BCa. In this study, this application analyzed the comprehensive pattern of DNA methylation and found that PENK methylation has high potential as a biomarker for urine-based early detection of BCa. Validation of PENK methylation confirmed that it can significantly improve the sensitivity and specificity of non-invasive detection of BCa. The kit provided in this application can detect the methylation status of TWIST1, NID2 and PENK genes related to bladder cancer in urine exfoliated cells by methylation-specific real-time fluorescence PCR, which is an auxiliary diagnostic method for initial diagnosis of bladder cancer.

[0084] The term "diagnosis" includes aspects of auxiliary diagnosis, recurrence risk assessment, cancer risk and degree of cancer assessment, prognosis judgment, etc.

[0085] The terms "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refer to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides can be produced in any manner, including chemical synthesis, DNA replication, reverse transcription, or combinations thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine, and guanine.

[0086] The term "methylation" is a form of chemical modification of DNA, which can change genetic expression without changing the sequence of DNA. DNA methylation refers to the covalent binding of a methyl group to the 5th carbon of cytosine in the CpG dinucleotide of the genome under the action of DNA methyltransferase. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and DNA-protein interaction mode, thereby controlling gene expression.

[0087] The term "methylation level" refers to whether methylation occurs in one or more CpG dinucleotides in a DNA sequence, or the frequency / proportion / percentage of methylation, which represents both qualitative and quantitative concepts.

[0088] In practical applications, different detection indicators can be used to compare the DNA methylation level according to the actual situation. For example, in some cases, the Ct value of the sample detection can be compared; in some cases, the proportion of gene methylation in the sample, i.e., the number of methylated molecules / (the number of methylated molecules + the number of non-methylated molecules) x 100, can be calculated, and then compared; in some cases, statistical analysis and integration of each indicator are also needed to obtain the final determination indicator.

[0089] The term "primer" refers to an oligonucleotide that can be used in an amplification method, such as polymerase chain reaction (PCR), to amplify a sequence of interest based on a polynucleotide sequence corresponding to a gene or a portion of a region thereof. Typically, at least one of the PCR primers used to amplify a polynucleotide sequence is sequence-specific for that polynucleotide sequence. The exact length of the primer depends on many factors, including temperature, source of the primer, and method used. For diagnostic and prognostic applications, for example, oligonucleotide primers typically contain at least 10, 15, 20, 25, or more nucleotides, but can contain fewer, depending on the complexity of the target sequence. In the present disclosure, the term "primer" or "primer pair" refers to a pair of primers that can hybridize to both strands of a target DNA molecule or can hybridize to regions flanking the nucleotide sequence to be amplified in a target DNA molecule.

[0090] The term "methylation-specific PCR" is one of the most sensitive experimental techniques for studying methylation, which can detect methylation of about 50 pg of DNA. After bisulfite conversion of single-stranded DNA, all unmethylated cytosines are deaminated to uracils, while methylated cytosines in CpG sites remain unchanged. Therefore, two pairs of primers are designed for methylated and unmethylated sequences, respectively, and the methylated and unmethylated DNA sequences can be distinguished by PCR amplification.

[0091] The term "methylation-specific fluorescent quantitative PCR (QMSP)" is an experimental technique combining the fluorescent quantitative PCR technique and the methylation-specific PCR technique. This technique is also based on the sequence difference of DNA with different methylation states after bisulfite conversion to design a suitable primer pair, thereby distinguishing the methylated and unmethylated sequences. However, the final detection index of q-MSP is the fluorescence signal, and therefore, in the q-MSP reaction system, in addition to the addition of methylation detection primers, a fluorescent probe or fluorescent dye also needs to be added. Compared with the traditional methylation-specific PCR technique, the sensitivity and specificity of q-MSP for detecting the methylation level of DNA are higher, and it is more suitable for detecting the trace amount of DNA fragments with abnormal methylation mixed in the DNA of early cancer patients. Moreover, this technique does not need gel electrophoresis detection, and the operation is more convenient.

[0092] The term "probe" refers to an oligonucleotide sequence containing a 5' fluorescent group and a 3' quenching group. When the probe binds to the corresponding site on the DNA, the probe does not emit fluorescence because there is a quenching group near the fluorescent group. During the amplification process, if the probe binds to the amplified strand, the 5'-3' exonuclease activity of DNA polymerase (such as Taq enzyme) will digest the probe, and the fluorescent group is away from the quenching group, and its energy is not absorbed, that is, a fluorescence signal is generated. Every time a PCR cycle is passed, the fluorescence signal also has a synchronous exponential growth process as the target fragment.

[0093] The "biological marker" in the present application refers to a substance such as a gene, a variable measurement related to a disease, which can be used as an indicator or predictor of that disease. The presence or risk of a disease can be inferred from the parameter of the biological marker without measuring the disease itself.

[0094] In a first aspect of the embodiments of the present application, a nucleic acid combination product is provided, which comprises a primer pair and a probe for detecting a biological marker;

[0095] The primer pair and the probe for detecting the biological marker comprise a primer pair and a probe for detecting the TWIST1 gene, and further comprise a primer pair and a probe for detecting one or more of the PENK gene and the NID2 gene;

[0096] The primer pair and probe for detecting the TWIST1 gene comprise a forward primer represented by any one of SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 10 and SEQ ID NO. 13, a reverse primer represented by any one of SEQ ID NO. 2, SEQ ID NO. 5, SEQ ID NO. 8, SEQ ID NO. 11 and SEQ ID NO. 14, and a probe represented by any one of SEQ ID NO. 3, SEQ ID NO. 6, SEQ ID NO. 9, SEQ ID NO. 12 and SEQ ID NO. 15;

[0097] The primer pair and probe for detecting the PENK gene comprise a forward primer represented by any one of SEQ ID NO. 16, SEQ ID NO. 19, SEQ ID NO. 22, SEQ ID NO. 25 and SEQ ID NO. 28, a reverse primer represented by any one of SEQ ID NO. 17, SEQ ID NO. 20, SEQ ID NO. 23, SEQ ID NO. 26 and SEQ ID NO. 29, and a probe represented by any one of SEQ ID NO. 18, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 27 and SEQ ID NO. 30;

[0098] The primer pair and probe for detecting the NID2 gene comprise a forward primer represented by any one of SEQ ID NO. 31 and SEQ ID NO. 34, a reverse primer represented by any one of SEQ ID NO. 32 and SEQ ID NO. 35, and a probe represented by any one of SEQ ID NO. 33 and SEQ ID NO. 36.

[0099] In some examples, the primer pair and probe for detecting the TWIST1 gene comprise a forward primer of SEQ ID NO. 1, a reverse primer of SEQ ID NO. 2, and a probe of SEQ ID NO. 3; or a forward primer of SEQ ID NO. 4, a reverse primer of SEQ ID NO. 5, and a probe of SEQ ID NO. 6; or a forward primer of SEQ ID NO. 7, a reverse primer of SEQ ID NO. 8, and a probe of SEQ ID NO. 9; or a forward primer of SEQ ID NO. 10, a reverse primer of SEQ ID NO. 11, and a probe of SEQ ID NO. 12; or a forward primer of SEQ ID NO. 13, a reverse primer of SEQ ID NO. 14, and a probe of SEQ ID NO. 15.

[0100] In some examples, the primer pair and probe for detecting the PENK gene comprise a forward primer of SEQ ID NO. 16, a reverse primer of SEQ ID NO. 17, and a probe of SEQ ID NO. 18; or a forward primer of SEQ ID NO. 19, a reverse primer of SEQ ID NO. 20, and a probe of SEQ ID NO. 21; or a forward primer of SEQ ID NO. 22, a reverse primer of SEQ ID NO. 23, and a probe of SEQ ID NO. 24; or a forward primer of SEQ ID NO. 25, a reverse primer of SEQ ID NO. 26, and a probe of SEQ ID NO. 27; or a forward primer of SEQ ID NO. 28, a reverse primer of SEQ ID NO. 29, and a probe of SEQ ID NO. 30.

[0101] In some examples, the primer pair and probe for detecting the NID2 gene comprise a forward primer of SEQ ID NO. 31, a reverse primer of SEQ ID NO. 32, and a probe of SEQ ID NO. 33; or a forward primer of SEQ ID NO. 34, a reverse primer of SEQ ID NO. 35, and a probe of SEQ ID NO. 36.

[0102] In some examples, the primer pair and probe for detecting the biomarker consist of the primer pair and probe for detecting the TWIST1 gene and the primer pair and probe for detecting the PENK gene. Optionally, the primer pair and probe for detecting the biomarker comprise Nucleic Acid Combination 5 and Nucleic Acid Combination 10, for example as described in Example 1.

[0103] In some examples, the primer pair and probe for detecting the biomarker consist of the primer pair and probe for detecting the TWIST1 gene and the primer pair and probe for detecting the NID2 gene. Optionally, the primer pair and probe for detecting the biomarker comprise Nucleic Acid Combination 5 and Nucleic Acid Combination 12, for example as described in Example 2.

[0104] The method for detecting using the nucleic acid combination product is not particularly limited in the present application, for example, the detection of the methylation level of the target region can be achieved by one or more of the following methods: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole genome bisulfite sequencing, pyrosequencing, methylation-specific high performance liquid chromatography, digital PCR, methylation-specific high resolution melting curve, methylation-sensitive restriction enzyme, and methylation-specific fluorescent quantitative PCR.

[0105] In some examples, the 5' end of the probe in the primer pair and probe for detecting the biomarker is labeled with a fluorescent group, and the 3' end is labeled with a quencher group; optionally, the excitation light waves of the fluorescent groups of the probes for detecting different biomarkers are different.

[0106] In some examples, the nucleic acid combination product further comprises a primer pair and probe for detecting a reference gene;

[0107] Optionally, the primer pair and probe for detecting the reference gene comprise a forward primer as shown in SEQ ID NO. 37, a reverse primer as shown in SEQ ID NO. 38, and a probe as shown in SEQ ID NO. 39;

[0108] Further optionally, the 5' end of the probe in the primer pair and probe for detecting the reference gene is labeled with a fluorescent group, and the 3' end is labeled with a quencher group;

[0109] More further, the probe in the primer pair and probe for detecting the reference gene and the probe in the primer pair and probe for detecting the biomarker have fluorescent groups with different excitation light waves.

[0110] In a second aspect of the embodiments of the present application, the nucleic acid combination product is provided for use in the preparation of a bladder cancer diagnostic kit or a urothelial carcinoma diagnostic kit.

[0111] In a third aspect, the present application provides a kit comprising the nucleic acid combination product.

[0112] In some examples, the kit further comprises one or more of a DNA amplification reagent, a reagent for converting unmethylated cytosine bases to uracil, a DNA extraction reagent, and a DNA purification reagent.

[0113] The nucleic acid combination product and the kit provided by the present application are suitable for the auxiliary diagnosis of patients suspected of having bladder cancer, patients recommended for cystoscopy, and patients with clinical symptoms such as hematuria, frequent urination, urgency, and pain, and can be used as a supplement and auxiliary to the existing diagnostic methods for the reference of clinicians.

[0114] The nucleic acid combination product and the kit provided by the present application are suitable for the auxiliary diagnosis of patients suspected of having urothelial carcinoma, patients with lower back pain, and patients with full-range hematuria accompanied by strip-shaped blood clots, and can be used as a supplement and auxiliary to the existing diagnostic methods for the reference of clinicians.

[0115] The nucleic acid combination and the kit provided by the present application can be used for detecting urine samples. In the present application, the "subject" or "patient" or "subject" includes human patients and other mammals, and also includes any individual who has or has bladder cancer, or any individual who wishes to use the method of the present application for analysis or treatment. Suitable mammals falling within the scope of the present application include, but are not limited to, primates, domestic animals (e.g., sheep, cattle, horses, monkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), pets (e.g., cats, dogs), and captive wild animals (e.g., foxes, deer, dingoes). Preferably, the patient is a human patient.

[0116] The following are embodiments of the nucleic acid combination product, the kit, and the method of detecting a urine sample using the nucleic acid combination product of the present application. It can be understood that, considering the general description provided above, a variety of other embodiments can be implemented.

[0117] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. In the following examples, the experimental methods not specified in the specific conditions can be referred to the guidelines provided in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0118] In the following specific examples, the measurement parameters of the raw material components may, without specific instructions, have slight deviations within the weighing accuracy range. With respect to temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0119] Table 1, primer pair and probe for detecting TWIST1 gene

[0120]

[0121] Table 2, primer pair and probe for detecting PENK gene

[0122]

[0123] Table 3, primer pair and probe for detecting NID2 gene

[0124]

[0125] Table 4, primer pair and probe for detecting GAPDH gene

[0126]

[0127] In Tables 1 to 4, the primer sequences are all from 5' end to 3' end.

[0128] Example 1

[0129] 1. Sample selection

[0130] 360 urine samples were selected, including 94 urine samples of bladder cancer patients, 114 urine samples of patients with interference diseases, and 152 urine samples of healthy people.

[0131] Sample inclusion criteria:

[0132] The following conditions were used for the inclusion of bladder cancer: The histological classification of bladder tumor is recommended to use the classification standard of WHO Classification of Tumors of the Urinary System and Male Genital Organs (4th edition) in 2016, which has some changes compared with the 2004 edition. The bladder cancer staging standard uses the eighth edition standard of AJCC 2017, according to the TNM staging system. Histopathological examination is used as the gold standard, and the final diagnosis is made by relying on the professionalism of experienced clinicians.

[0133] Interference sample inclusion: Patients with diagnosed prostate hyperplasia, bladder stones, and urothelial infection were included, and the pathological results were negative to exclude bladder cancer.

[0134] Three conditions were used for the selection of the healthy group: (1) According to the questionnaire, subjects with common chronic diseases (hypertension, diabetes, coronary heart disease), history of tumor treatment, and major surgery were excluded; (2) No obvious clinical symptoms were reported; (3) The results of physical examination were within the normal value range and no obvious abnormalities were found; the examination results were outside the normal value range or had abnormalities but were judged by the doctor to have no clinical significance. (The physical examination items include: LDCT, abdominal color Doppler ultrasound, 4 tumor markers, blood pressure, etc., as shown in the physical examination items).

[0135] Exclusion criteria: under 18 years old; pregnant; duplicate samples, undiagnosed, or unqualified quality inspection.

[0136] 2. DNA Extraction

[0137] Using Quick-DNA TM Urine Kit Catalog No. D3061 (ZYMO RESEARCH) easily purifies cellular DNA / cell-free DNA from up to 40 mL of urine. DNA yield - The DNA binding capacity of the column is 5 μg.

[0138] Please note that DNA yields may vary depending on the urine itself. Female urine generally yields more DNA than male urine. The average urine DNA amount in healthy female individuals ranges from 6 ng / mL to 1000 ng / mL. The average DNA amount in healthy male individuals ranges from 2 to 20 ng / mL.

[0139] 3. Methylation detection

[0140] The extracted and purified DNA / cell-free DNA was treated with bisulfite and purified and recovered using the ZYMO Methylation Kit D5005. For specific steps, refer to the kit's instructions. 20 μL of Bis-DNA was ultimately obtained from each urine sample.

[0141] Using Bis-DNA as a template, PCR reaction systems for TWIST1, PENK, and the internal reference gene GAPDH were prepared according to Table 5. The primer pairs and probes used included nucleic acid combination 5 in Table 1 (TWIST1-F5: SEQ ID NO. 13; TWIST1-R5: SEQ ID NO. 14; TWIST1-P5: SEQ ID NO. 15), nucleic acid combination 10 in Table 2 (PENK-F4: SEQ ID NO. 28; PENK-R4: SEQ ID NO. 29; PENK-P4: SEQ ID NO. 30), and nucleic acid combination 13 in Table 4.

[0142] Table 5. Reaction system

[0143] Ingredients Amount 2x qPCR Mix (containing taq enzyme, reaction buffer, dNTP, etc.) 12.5 μL Forward primer (10 μM) 0.5 μL Reaction primer (10 μM) 0.5 μL Probe (10 μM) 0.5 μL Nuclease-free water 6 μL Bis-DNA (amplification template, concentration 0.2 ng / μL) 5.0 μL Total volume 25 μL

[0144] Table 6. Reaction conditions and instruments

[0145]

[0146] Standard references: The positive control is bladder cancer tissue DNA; the negative control is leukocyte DNA. The reaction systems and conditions for the corresponding controls are as shown in Tables 5 and 6.

[0147] 4. Judgment basis

[0148] The Ct values of the internal reference gene, the TWIST1 gene, and the PENK gene are represented by Ct0, Ct1, and Ct2, respectively, ΔCt1 = |Ct1-Ct0|, and ΔCt2 = |Ct2-Ct0|. A Ct0 value greater than 35 is considered to be unqualified for detection. The results are determined under the condition that the Ct0 value is less than or equal to 35.

[0149] Table 7. Judgment basis for combined detection of the TWIST1 gene and the PENK gene

[0150]

[0151] Under the condition of single PENK gene detection, the following judgment basis is used: Ct2≤35 and ΔCt2<8, which is determined as positive; otherwise, it is determined as negative.

[0152] Table 8. Experimental results of combined detection of the TWIST1 gene and the PENK gene

[0153]

[0154] The sensitivity is the proportion of samples with positive methylation detection results among samples with positive pathological results; the specificity is the proportion of samples with negative methylation detection results among samples with negative pathological results; and the accuracy is the proportion of samples with detection results among samples. The ROC curve is shown in Figure 1 .

[0155] Example 2

[0156] Use case of combination of the TWIST1 gene and the NID2 gene.

[0157] 1. Sample selection The same as in Example 1.

[0158] 2. DNA extraction The same as in Example 1.

[0159] 3. Methylation detection Basically the same as in Example 1, with the difference that the nucleic acid combination 5 in Table 1, the nucleic acid combination 13 in Table 4, and the nucleic acid combination 12 (NID2-F2: SEQ ID NO. 34, NID2-R2: SEQ ID NO. 35, NID2-P2: SEQ ID NO. 36) in Table 3 are used in combination.

[0160] 4. Judgment basis

[0161] The Ct values of the internal reference gene, TWIST1 gene, and NID2 gene are represented by Ct0, Ct1, and Ct3, respectively, ΔCt1 = |Ct1-Ct0|, ΔCt3 = |Ct3-Ct0|, and a Ct0 value greater than 35 is regarded as unqualified for detection. The result determination is performed under the condition that the Ct0 value is less than or equal to 35.

[0162] (1) The determination basis corresponding to single TWIST1 is: Ct1≤35, which is determined as positive; otherwise, it is determined as negative.

[0163] (2) The determination basis corresponding to single NID2 is: Ct3≤35, which is determined as positive; otherwise, it is determined as negative.

[0164] (3) The determination basis of combined detection of TWIST1 and NID2 is:

[0165] 1) Ct1≤35, without the need to determine NID2, it can be determined as positive;

[0166] 2) 35

[0167] 1) and 2) are determined as negative.

[0168] 5、Results

[0169] Table 9, Experimental results of combined detection of TWIST1 gene and NID2 gene

[0170] Single TWIST1 Single NID2 TWIST1, NID2 combination Out of 94 patients 28 negatives 46 negatives 10 negatives Out of 114 interferers 0 positives 4 positives 12 positives Out of 152 healthy people 4 positives 2 positives 4 positives

[0171] The results in the table show that the sensitivity of single TWIST1 detection is 70.21%, the specificity is 98.496%, and the accuracy is 91.11%; the sensitivity of single NID2 detection is 51.06%, the specificity is 97.74%, and the accuracy is 85.56%; the sensitivity of combined detection of TWIST1 and NID2 is 89.36%, the specificity is 93.98%, and the accuracy is 92.78%. The ROC curve is shown in Figure 2 .

[0172] Example 3

[0173] This example is a blind test example, a total of 220 urine samples, the pathological condition of the urine samples is unknown (the hospital knows the pathological condition), the results after detection are submitted to the hospital, and the hospital will give the coincidence rate of the experimental results for feedback.

[0174] The experiment is performed in the same way as in Example 1, and the final determination result is shown in Table 10.

[0175] In 220 samples: 74 samples were positive in 78 positive samples, 4 were negative; 4 samples were detected as positive in 70 interference samples; 2 samples were detected as positive in 72 healthy human samples.

[0176] Table 10

[0177]

[0178] The positive control (bladder cancer tissue DNA) was detected by using the nucleic acid combination in Table 1, the reaction system described in Table 5 in Example 1, the reaction conditions and the instrument described in Table 6, and the results are shown in Table 7. Figure 3 (corresponding to nucleic acid combination 1 in Table 1), Figure 4 (corresponding to nucleic acid combination 2 in Table 1), Figure 5 (corresponding to nucleic acid combination 3 in Table 1), Figure 6 (corresponding to nucleic acid combination 4 in Table 1), Figure 7 (corresponding to nucleic acid combination 5 in Table 1). According to the detection results, the effect of nucleic acid combination 5 in Table 1 is the best.

[0179] The positive control (bladder cancer tissue DNA) was detected by using the nucleic acid combination in Table 2, the reaction system described in Table 5 in Example 1, the reaction conditions and the instrument described in Table 6, and the results are shown in Table 8. Figure 8 (corresponding to nucleic acid combination 6 in Table 2), Figure 9 (corresponding to nucleic acid combination 7 in Table 2), Figure 10 (corresponding to nucleic acid combination 8 in Table 2), Figure 11 (corresponding to nucleic acid combination 9 in Table 2), Figure 12 (corresponding to nucleic acid combination 10 in Table 2). According to the detection results, the effect of nucleic acid combination 10 in Table 2 is the best.

[0180] The positive control (bladder cancer tissue DNA) was detected by using the nucleic acid combination in Table 3, the reaction system described in Table 5 in Example 1, the reaction conditions and the instrument described in Table 6, and the results are shown in Table 9. Figure 13 (corresponding to nucleic acid combination 11 in Table 3), Figure 14 (corresponding to nucleic acid combination 12 in Table 3). According to the detection results, the effect of nucleic acid combination 12 in Table 3 is the best.

[0181] The positive control (bladder cancer tissue DNA) was detected by using the nucleic acid combination 5 and the nucleic acid combination 10, the reaction system described in Table 5 in Example 1, the reaction conditions and the instrument described in Table 6, and the results are shown in Table 10. Figure 15 Pink is the internal reference gene, blue is the detection result of PENK gene, and green is the detection result of TWIST1 gene. This is the result of TWIST1 and PENK combined detection displayed on the same graph.

[0182] The reaction system described in Table 5 in Example 1, the reaction conditions described in Table 6, and the instrument were used to detect the positive control (bladder cancer tissue DNA) using nucleic acid combination 5 and nucleic acid combination 12, and the results are shown in FIG. 6. Figure 16 , the pink color is the internal reference gene, the blue color is the NID2 gene detection result, and the green color is the TWIST1 gene detection result. This is the result of the combined detection of TWIST1 and NID2, which is shown on the same graph.

[0183] Overall, the embodiments of the present application provide a nucleic acid combination product and a kit for detecting the TWIST1 gene and jointly detecting at least one of the NID2 and PENK genes, thereby enabling efficient and sensitive diagnosis and detection of cancer in a non-invasive manner.

[0184] Example 4

[0185] 1. Sample selection

[0186] 370 urine samples were selected, including 104 urine samples from patients with urothelial carcinoma, 114 urine samples from patients with interference diseases, and 152 urine samples from healthy people.

[0187] Sample inclusion criteria:

[0188] The following conditions were used for the inclusion of urothelial carcinoma patients: patients diagnosed with urothelial carcinoma by clinical diagnosis or case diagnosis;

[0189] Interference sample inclusion: subjects diagnosed with benign diseases of the urinary system by clinical diagnosis;

[0190] Three conditions were used for the selection of the healthy group: (1) subjects with common chronic diseases (hypertension, diabetes, coronary heart disease), history of tumor treatment, and major surgery were excluded according to the questionnaire; (2) subjects who reported no obvious clinical symptoms; (3) subjects whose physical examination results were within the normal range and showed no obvious abnormalities; subjects whose test results were outside the normal range or showed abnormalities but were judged by the doctor to have no clinical significance (the physical examination items included: LDCT, abdominal ultrasound, 4 tumor markers, blood pressure, etc., as shown in the figure).

[0191] Exclusion criteria: under 18 years old; pregnant; sample duplication, not diagnosed, and unqualified quality inspection.

[0192] 2. DNA extraction: same as Example 1.

[0193] 3. Methylation detection: same as Example 1.

[0194] 4. Judgment basis: same as Example 1.

[0195] 5. Detection results

[0196] Table 11. Experimental results for detection of urothelial carcinoma

[0197]

[0198] Each technical feature of the above-described embodiments and examples can be combined in any suitable manner, and for brevity, not all possible combinations are described, but it is to be understood that the scope of protection includes all possible combinations.

[0199] The above-described embodiments are merely representative of several embodiments of the present application, and facilitate a specific and detailed understanding of the technical solutions of the present application, but should not be construed as limiting the scope of patent protection. It should be noted that, for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the scope of protection of the present application. In addition, it should be understood that, after reading the above teachings of the present application, those skilled in the art can make various modifications or improvements to the present application, and the equivalent forms obtained are also within the scope of protection of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art can obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the scope of protection of the appended claims of the present application. Therefore, the scope of protection of the patent of the present application should be based on the contents of the appended claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. Nucleic acid combination products, which include primer pairs and probes for detecting biomarkers; The primer pair and probe for detecting biomarkers are: a primer pair and probe for detecting the TWIST1 gene, and a primer pair and probe for detecting the PENK gene; The primer pair and probe for detecting the TWIST1 gene are: a forward primer shown in SEQ ID NO.13, a reverse primer shown in SEQ ID NO.14, and a probe shown in SEQ ID NO.15; The primer pair and probe for detecting the PENK gene are: the forward primer shown in SEQ ID NO.28, the reverse primer shown in SEQ ID NO.29, and the probe shown in SEQ ID NO.

30.

2. The nucleic acid combination product according to claim 1, wherein In the primer pair and probe for detecting biomarkers, the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

3. The nucleic acid combination product according to claim 2, wherein The fluorescent groups of the probes detecting different biomarkers have different excitation light wavelengths.

4. The nucleic acid combination product according to any one of claims 1 and 3, further comprising a primer pair and a probe for detecting an internal reference gene.

5. The nucleic acid combination product according to claim 4, wherein The primer pair and probe for detecting the internal reference gene include the forward primer shown in SEQ ID NO.37, the reverse primer shown in SEQ ID NO.38, and the probe shown in SEQ ID NO.

39.

6. The nucleic acid combination product according to claim 5, wherein In the primer pair and probe for detecting the internal reference gene, the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

7. The nucleic acid combination product according to claim 6, wherein The probe in the primer pair and probe for detecting the internal reference gene and the probe in the primer pair and probe for detecting the biomarker have fluorescent groups with different excitation light waves.

8. Use of the nucleic acid combination product according to any one of claims 1 to 7 in the preparation of a bladder cancer diagnostic kit.

9. A kit comprising the nucleic acid combination product according to any one of claims 1 to 7.

10. The kit according to claim 9, further comprising one or more of a DNA amplification reagent, a reagent for converting unmethylated cytosine bases into uracil, a DNA extraction reagent, and a DNA purification reagent.

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