Reagent for detecting methylation level of molecular marker and urinary tract epithelial cancer detection kit

Abstract

The application relates to a reagent for detecting the methylation level of a molecular marker and a urinary epithelial carcinoma detection kit. By detecting the methylation level of six molecular markers in a sample of a subject, cancer patients and non-cancer patients can be effectively distinguished, the urinary epithelial carcinoma can be diagnosed, and high sensitivity and specificity are achieved.

Description

Technical Field

[0001] This application relates to the field of molecular biology, and in particular to reagents for detecting the methylation level of molecular markers and kits for detecting urothelial carcinoma. Background Technology

[0002] Urothelial carcinoma (UC) is a group of multifocal malignant tumors originating from the urothelial tract, including renal pelvis cancer, ureteral cancer, bladder cancer, and urethral cancer. It is the most common urinary system tumor, with bladder cancer accounting for over 90% of all urothelial carcinomas. Only about 5%–10% of cases are upper urinary tract urothelial carcinoma. Approximately 8%–13% of upper urinary tract urothelial carcinoma cases also have bladder urothelial carcinoma. Statistics show that in 2018, there were approximately 550,000 new cases of bladder urothelial carcinoma worldwide, with about 200,000 deaths. Approximately 25% of patients are diagnosed with muscle-invasive or metastatic bladder cancer at the time of initial diagnosis. In addition, about 30% to 40% of non-muscle-invasive bladder cancers will gradually invade the bladder wall and progress to muscle-invasive cancer, resulting in a poor prognosis and a high mortality rate. For example, the 5-year survival rate of patients with muscle-invasive bladder cancer is about 30% to 50%.

[0003] Currently, commonly used clinical methods for diagnosing bladder cancer include cystoscopy, urine cytology, fluorescence in situ hybridization (FISH), and imaging. Cystoscopy and pathological biopsy are the gold standard for diagnosing bladder cancer; however, as invasive procedures, they carry risks such as urethral perforation and urethral infection, leading to low patient acceptance. Urine cytology and FISH are non-invasive procedures with good specificity for diagnosing bladder cancer, but both methods have low sensitivity for early-stage non-muscle-invasive cancer. Imaging techniques such as CT scans are also non-invasive, but they involve radiation and are not suitable for large-scale screening. Therefore, there is an urgent need for non-invasive, convenient, and highly sensitive methods for diagnosing bladder cancer and upper urinary tract urothelial carcinoma.

[0004] DNA methylation is an epigenetic modification pattern in the human genome. Abnormal DNA methylation has been reported to be associated with cancer and other diseases by regulating genomic transcription levels. For example, in highly repetitive DNA sequences, hypomethylation leads to chromosomal instability, thereby activating oncogenes; while hypermethylation in gene promoter regions leads to transcriptional silencing of some tumor suppressor genes. Evidence suggests that abnormal DNA methylation is a major characteristic of bladder cancer, playing a crucial role in the occurrence and development of bladder tumors. This altered methylation pattern is stable and easily detectable in bodily fluid samples such as blood. Therefore, genes exhibiting abnormal methylation can be used as biomarkers for the early diagnosis and prognosis of bladder cancer. Summary of the Invention

[0005] Therefore, it is necessary to provide a reagent for detecting the methylation level of molecular markers and its application in the preparation of a detection product for urothelial carcinoma, which has high sensitivity and specificity for the diagnosis of very early and early-stage cancers.

[0006] The specific technical solution is as follows:

[0007] A reagent for detecting the methylation level of a molecular marker, said molecular marker comprising at least one of BM1, BM2, BM3, BM4, BM5, and BM6 as defined below:

[0008] Using GRCh38.p14 as the reference genome, BM1 is the full-length or partial region of Chr10:101140226-101140445, BM2 is the full-length or partial region of Chr10:116271316-116271587, BM3 is the full-length or partial region of Chr13:27929099-27929475, BM4 is the full-length or partial region of Chr6:27495249-27495644, BM5 is the full-length or partial region of Chr7:19118066-19118518, and BM6 is the full-length or partial region of Chr10:17229173-17229587.

[0009] In one embodiment, BM1 includes at least one of BM1-1, BM1-2, BM1-3, BM1-4, BM1-5, and BM1-6 as defined below, wherein BM1-1 is a positive chain of Chr10:101140255-101140395, BM1-2 is a positive chain of Chr10:101140325-101140440, BM1-3 is a positive chain of Chr10:101140237-101140445, BM1-4 is a negative chain of Chr10:101140400-101140234, and BM1-5 is a negative chain of Chr10:101140385-10114. 0254 is a negative chain, wherein BM1-6 is a Chr10:101140374-101140226 negative chain; BM2 includes at least one of BM2-1, BM2-2 and BM2-3 as defined below, wherein BM2-1 is a Chr10:116271316-116271435 positive chain, wherein BM2-2 is a Chr10:116271441-116271537 positive chain, and BM2-3 is a Chr10:116271440-116271587 positive chain; BM3 includes at least one of BM3-1, BM3-2, BM3-3 and BM3-4 as defined below, wherein BM3-1 is a C hr13:27929099-27929205 is a positive chain, BM3-2 is a positive chain of Chr13:27929219-27929389, BM3-3 is a negative chain of Chr13:27929475-27929301, and BM3-4 is a negative chain of Chr13:27929259-27929146; BM4 includes at least one of BM4-1, BM4-2, BM4-3, BM4-4, and BM4-5 as defined below, where BM4-1 is a positive chain of Chr6:27495249-27495413, and BM4-2 is a positive chain of Chr6:27495394-2749550. 3 positive chains, wherein BM4-3 is a positive chain of Chr6:27495524-27495644, BM4-4 is a negative chain of Chr6:27495603-27495461, and BM4-5 is a negative chain of Chr6:27495445-27495376; BM5 includes at least one of BM5-1, BM5-2, and BM5-3 as defined below, wherein BM5-1 is a positive chain of Chr7:19118066-19118214, BM5-2 is a positive chain of Chr7:19118242-19118334, and BM5-3 is a positive chain of Chr7:19118399-19118518;And / or, BM6 includes at least one of BM6-1, BM6-2, BM6-3, BM6-4, BM6-6 as defined below, wherein BM6-1 is a positive chain of Chr10:17229173-17229323, BM6-2 is a positive chain of Chr10:17229290-17229401, BM6-3 is a positive chain of Chr10:17229424-17229571, BM6-4 is a negative chain of Chr10:17229587-17229450, BM6-5 is a negative chain of Chr10:17229437-17229291, and BM6-6 is a negative chain of Chr10:17229305-17229173.

[0010] In one embodiment, the molecular markers include at least any two of BM1, BM4, and BM6.

[0011] In one embodiment, the reagent includes primer pairs and / or probes capable of detecting the methylation level of the molecular marker.

[0012] In one embodiment, the reagent includes primer pairs for detecting the methylation level of BM1, wherein the primer pairs include at least one of the primer pairs shown in SEQ ID NO. 19-20, SEQ ID NO. 22-23, SEQ ID NO. 25-26, SEQ ID NO. 28-29, SEQ ID NO. 31-32, or SEQ ID NO. 34-35; the reagent includes primer pairs for detecting BM2, wherein the primer pairs include at least one of the primer pairs shown in SEQ ID NO. 37-38, SEQ ID NO. 40-41, or SEQ ID NO. 43-44; the reagent includes primer pairs for detecting BM3, wherein the primer pairs include at least one of the primer pairs shown in SEQ ID NO. 46-47, SEQ ID NO. 49-50, SEQ ID NO. 52-53, or SEQ ID NO. 34-35. The reagent includes at least one of the primer pairs shown in SEQ ID NO. 55-56; the reagent includes a primer pair for detecting BM4, wherein the primer pair includes at least one of the primer pairs shown in SEQ ID NO. 58-59, SEQ ID NO. 61-62, SEQ ID NO. 64-65, SEQ ID NO. 67-68, or SEQ ID NO. 70-71; the reagent includes a primer pair for detecting BM5, wherein the primer pair includes at least one of the primer pairs shown in SEQ ID NO. 73-74, SEQ ID NO. 76-77, or SEQ ID NO. 79-80; and / or, the reagent includes a primer pair for detecting BM6, wherein the primer pair includes the primer pairs shown in SEQ ID NO. 82-83, SEQ ID NO. 85-86, SEQ ID NO. 88-89, SEQ ID NO. 70-71, SEQ ID NO. 70-71. At least one of the primer pairs shown in ID NO. 91–92, SEQ ID NO. 94–95, or SEQ ID NO. 97–98.

[0013] In one embodiment, the reagent includes a probe for detecting the methylation level of BM1, the probe including at least one of the probes shown in SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 27, SEQ ID NO. 30, SEQ ID NO. 33, or SEQ ID NO. 36; the reagent includes a probe for detecting the methylation level of BM2, the probe including at least one of the probes shown in SEQ ID NO. 39, SEQ ID NO. 42, or SEQ ID NO. 45; the reagent includes a probe for detecting the methylation level of BM3, the probe including at least one of the probes shown in SEQ ID NO. 48, SEQ ID NO. 51, SEQ ID NO. 54, or SEQ ID NO. 57; the reagent includes a probe for detecting the methylation level of BM4, the probe including at least one of the probes shown in SEQ ID NO. 60, SEQ ID NO. 63, SEQ ID NO. 66, SEQ ID NO. 69, or SEQ ID NO. 72; the reagent includes a probe for detecting the methylation level of BM5, the probe including at least one of the probes shown in SEQ ID NO. 75, SEQ ID NO. 60, SEQ ID NO. 63, SEQ ID NO. 66, SEQ ID NO. 69, or SEQ ID NO. 72; and the reagent includes a probe for detecting the methylation level of BM5, the probe including at least one of the probes shown in SEQ ID NO. 75, SEQ ID NO. 60, SEQ ID NO. 63, SEQ ID NO. 66, SEQ ID NO. 69, or SEQ ID NO. 72. The reagent comprises at least one of the probes shown in NO. 78 or SEQ ID NO. 81; and / or, the reagent comprises a probe for detecting the methylation level of the BM6, the probe comprising at least one of SEQ ID NO. 84, SEQ ID NO. 87, SEQ ID NO. 90, SEQ ID NO. 93, SEQ ID NO. 96, or SEQ ID NO. 99.

[0014] The above-mentioned reagents are used in the preparation of urothelial carcinoma detection products.

[0015] In one embodiment, the urothelial carcinoma includes bladder cancer and / or upper urinary tract urothelial carcinoma; alternatively, the upper urinary tract urothelial carcinoma includes ureteral cancer and renal pelvis cancer.

[0016] A diagnostic kit for urothelial carcinoma, the kit comprising the reagents described in any of the preceding claims.

[0017] In one embodiment, the test kit further includes at least one of a DNA bisulfite conversion reagent, a nucleic acid extraction reagent, a nucleic acid purification reagent, a PCR amplification reagent, and a quality control reagent. Optionally, the test kit further includes a sample collection device.

[0018] Compared with the prior art, this application has the following beneficial effects:

[0019] This application demonstrates an effective differentiation between bladder cancer patients and non-bladder cancer patients by modifying the methylation levels of six molecular markers in subject samples. Furthermore, detecting the methylation levels of a combination of markers shows higher sensitivity in diagnosing bladder cancer, ureteral cancer, and renal pelvis cancer, without a significant decrease in specificity. When the combination of markers is used as the target region, it also exhibits a high detection rate for very early and early-stage urothelial carcinoma, which is more conducive to the early diagnosis and treatment of urothelial carcinoma, improving patient survival rates and quality of life, bringing benefits to patients. Attached Figure Description

[0020] Figure 1 ROC curves of urine samples from patients with bladder cancer and healthy individuals diagnosed with BM1-1;

[0021] Figure 2 ROC curves of urine samples from patients with bladder cancer and healthy individuals using BM2-2 for diagnosis;

[0022] Figure 3 ROC curves of urine samples from patients with bladder cancer and healthy individuals diagnosed with BM3-4;

[0023] Figure 4 ROC curves of urine samples from patients with BM4-5 diagnosing bladder cancer and healthy individuals;

[0024] Figure 5 ROC curves of urine samples from patients with bladder cancer and healthy individuals using BM5-2 for diagnosis;

[0025] Figure 6 ROC curves of urine samples from patients with bladder cancer and healthy individuals diagnosed with BM6-1. Detailed Implementation

[0026] To facilitate understanding of this application, a more complete description will be provided below. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the disclosure of this application will be more thorough and complete.

[0027] 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 herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0028] Terminology Explanation

[0029] The term "and / or" refers to any and all combinations of one or more of the related listed items.

[0030] The term "multiple" refers to two or more; "various kinds" refers to two or more kinds; when "more than" is combined with a number to indicate quantity, it includes the number itself, for example, "two or more" includes two.

[0031] The term "diagnosis" includes aspects such as auxiliary diagnosis, recurrence risk assessment, assessment of cancer risk and degree, and prognosis.

[0032] The terms "oligonucleotide," "polynucleotide," "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 final function or use of the oligonucleotide. Oligonucleotides can be produced in any way, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine, and guanine.

[0033] The term "methylation" is a form of DNA chemical modification that can alter genetic expression without changing the DNA sequence. DNA methylation refers to the covalent binding of a methyl group to the 5th carbon position of cytosine in a CpG dinucleotide of the genome, under the action of DNA methyltransferases. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression.

[0034] The term "methylation level" refers to whether cytosine in one or more CpG dinucleotides within a DNA sequence is methylated, or the frequency / proportion / percentage of methylation. It represents both a qualitative and quantitative concept. In practical applications, different detection indicators can be used to compare DNA methylation levels depending on the specific circumstances. For example, in some cases, comparisons can be made based on the Ct values ​​of the samples; in others, the proportion of gene methylation in the sample can be calculated as (number of methylated molecules / (number of methylated molecules + number of unmethylated molecules)) × 100%, and then compared; in still others, statistical analysis and integration of various indicators are necessary to arrive at a final judgment criterion.

[0035] The term "primer" refers to an oligonucleotide that can be used in amplification methods (such as polymerase chain reaction PCR) to amplify a target sequence based on a polynucleotide sequence corresponding to a target gene or a region thereof. Typically, at least one of the PCR primers used to amplify a polynucleotide sequence is sequence-specific to that polynucleotide sequence. The exact length of a primer depends on many factors, including temperature, primer source, and the method used. For example, for diagnostic and prognostic applications, oligonucleotide primers typically contain at least 10, 15, 20, 25, or more nucleotides, depending on the complexity of the target sequence, but may also contain fewer nucleotides. In this disclosure, the term "primer" refers to a pair of primers capable of hybridizing to the double strand of a target DNA molecule or to regions of the target DNA molecule located on either side of the nucleotide sequence to be amplified.

[0036] The term "methylation-specific PCR" is one of the most sensitive experimental techniques for studying methylation, capable of detecting methylation in as little as approximately 50 pg of DNA. After single-stranded DNA undergoes bisulfite conversion, all unmethylated cytosine is deaminated to uracil, while methylated cytosine at CpG sites remains unchanged. Therefore, two pairs of primers are designed, one for methylated and one for unmethylated sequences, and PCR amplification can distinguish between methylated and unmethylated DNA sequences. In this disclosure, methylation primers are added during real-time quantitative methylation-specific PCR. If the Ct value meets the aforementioned requirements (e.g., Ct ≤ 38 in tissue samples), it indicates that the target sequence is methylated.

[0037] The term "methylation-specific quantitative PCR (q-MSP)" is an experimental technique that combines quantitative real-time PCR (qPCR) with methylation-specific PCR (MSP). This technique also uses the sequence differences resulting from bisulfite conversion of DNA in different methylation states to design appropriate primer pairs, thereby distinguishing between methylated and unmethylated sequences. However, the final detection indicator in q-MSP is the fluorescence signal; therefore, in addition to adding methylation detection primers, fluorescent probes or fluorescent dyes are also required in the q-MSP reaction system. Compared to traditional methylation-specific PCR, q-MSP offers higher sensitivity and specificity for detecting DNA methylation levels, making it more suitable for detecting trace amounts of abnormally methylated DNA fragments mixed in the DNA of early-stage cancer patients. Furthermore, this technique does not require gel electrophoresis, making it simpler to operate.

[0038] The term "TaqMan probe" refers to an oligonucleotide sequence containing a 5' fluorescent group and a 3' quencher group. When the probe binds to the corresponding site on DNA, it does not fluoresce because of the presence of the quencher group near the fluorescent group. During amplification, if the probe binds to the strand being amplified, the 5'-3' exonuclease activity of a DNA polymerase (such as Taq polymerase) digests the probe. Since the fluorescent group is far from the quencher group, its energy is not absorbed, thus producing a fluorescent signal. With each PCR cycle, the fluorescence signal, like the target fragment, undergoes a synchronous exponential growth process.

[0039] The term "AUC" is an abbreviation for "Area Under the Curve." Specifically, it refers to the area under the Receiver Operating Characteristic (ROC) curve. An ROC curve is a graph comparing the true positive rate to the false positive rate at different possible cut-off points of a diagnostic test. It depends on the trade-off between sensitivity and specificity at the chosen cut-off point (any increase in sensitivity will be accompanied by a decrease in specificity). The area under the ROC curve (AUC) is a measure of the accuracy of a diagnostic test (a larger area is better; the optimal value is 1; randomized tests will have an ROC curve located diagonally with an area of ​​0.5).

[0040] During their research, the inventors discovered that six DNA regions can serve as molecular markers for diagnosing urothelial carcinoma. By detecting changes in the methylation levels of these six molecular markers in tissue and urine samples from subjects, they can effectively distinguish between bladder cancer patients and healthy individuals. The detection exhibits high sensitivity and specificity. When simultaneously detecting the methylation levels of any two markers from BM1, BM4, and BM6, the sensitivity for diagnosing bladder, ureteral, and renal pelvis cancer is even higher, without a significant decrease in specificity. When using any two markers from BM1, BM4, and BM6 as the target region, it also shows a high detection rate for very early and early-stage urothelial carcinoma, further facilitating early diagnosis and treatment of urothelial carcinoma.

[0041] First, one embodiment of this application provides a reagent for detecting the methylation level of a molecular marker, wherein the molecular marker includes at least one of BM1, BM2, BM3, BM4, BM5, and BM6 as defined below:

[0042] BM1 is the entire or part of the region of Chr10:101140226-101140445; BM2 is the entire or part of the region of Chr10:116271316-116271587; BM3 is the entire or part of the region of Chr13:27929099-27929475; BM4 is the entire or part of the region of Chr6:27495249-27495644; BM5 is the entire or part of the region of Chr7:19118066-19118518; and BM6 is the entire or part of the region of Chr10:17229173-17229587.

[0043] It should be noted that, unless otherwise specified, all positions on chromosomes in this article refer to GRCh38.p14. Furthermore, because chromosomal DNA is a double-stranded structure composed of positive and negative strands, for regions represented by chromosomal positions, if it is not specified whether it represents the positive or negative strand of DNA, it means that it could be the positive strand, the negative strand, or both strands of DNA in that region. For example, BM1 is Chr10:101140226-101140445, which means that BM1 could be the positive strand of DNA within the Chr10:101140226-101140445 region, the negative strand of DNA within the Chr10:101140226-101140445 region, or both strands of DNA within the Chr10:101140226-101140445 region.

[0044] A portion of BM1 comprises DNA fragments physically located between Chr10:101140226 and 101140445. In a specific example, BM1 includes at least one of BM1-1, BM1-2, BM1-3, BM1-4, BM1-5, and BM1-6 as defined below. BM1-1 is a positive strand of Chr10:101140255-101140395; BM1-2 is a positive strand of Chr10:101140325-101140440; BM1-3 is a positive strand of Chr10:101140237-101140445; BM1-4 is a negative strand of Chr10:101140400-101140234; BM1-5 is a negative strand of Chr10:101140385-101140254; and BM1-6 is a negative strand of Chr10:101140374-101140226. It is understandable that parts of BM1 also include other unlisted DNA fragments located between Chr10:101140226 and 101140445.

[0045] A portion of BM2 includes DNA fragments physically located between Chr10:116271316 and 116271587. In a specific example, BM2 includes at least one of BM2-1, BM2-2, and BM2-3 as defined below: BM2-1 is a positive strand of Chr10:116271316-116271435, BM2-2 is a positive strand of Chr10:116271441-116271537, and BM2-3 is a positive strand of Chr10:116271440-116271587. It is understood that a portion of BM2 also includes other unlisted DNA fragments located between Chr10:116271316 and 116271587.

[0046] A portion of BM3 includes DNA fragments physically located between Chr13:27929099 and 27929475. In a specific example, BM3 includes at least one of BM3-1, BM3-2, BM3-3, and BM3-4 as defined below: BM3-1 is a positive strand of Chr13:27929099-27929205; BM3-2 is a positive strand of Chr13:27929219-27929389; BM3-3 is a negative strand of Chr13:27929475-27929301; and BM3-4 is a negative strand of Chr13:27929259-27929146. It is understood that a portion of BM3 also includes other unlisted DNA fragments located between Chr13:27929099 and 27929475.

[0047] A portion of BM4 comprises a DNA fragment physically located between Chr6:27495249 and 27495644. In a specific example, BM4 includes at least one of BM4-1, BM4-2, BM4-3, BM4-4, and BM4-5 as defined below: BM4-1 is a positive strand of Chr6:27495249-27495413; BM4-2 is a positive strand of Chr6:27495394-27495503; BM4-3 is a positive strand of Chr6:27495524-27495644; BM4-4 is a negative strand of Chr6:27495603-27495461; and BM4-5 is a negative strand of Chr6:27495445-27495376. Understandably, parts of BM4 also include other unlisted DNA fragments located between Chr6:27495249 and 27495644.

[0048] A portion of BM5 comprises DNA fragments physically located between Chr7:19118066 and 19118518. In a specific example, BM5 includes at least one of BM5-1, BM5-2, and BM5-3 as defined below: BM5-1 is a positive strand of Chr7:19118066-19118214, BM5-2 is a positive strand of Chr7:19118242-19118334, and BM5-3 is a positive strand of Chr7:19118399-19118518. It is understood that a portion of BM5 may also include other unlisted DNA fragments located between Chr7:19118066 and 19118518.

[0049] Part of BM6 includes a DNA fragment physically located between Chr10:17229173 and 17229587. In a specific example, BM6 includes at least one of BM6-1, BM6-2, BM6-3, BM6-4, BM6-6 as defined below, where BM6-1 is a positive chain of Chr10:17229173-17229323, BM6-2 is a positive chain of Chr10:17229290-17229401, BM6-3 is a positive chain of Chr10:17229424-17229571, BM6-4 is a negative chain of Chr10:17229587-17229450, BM6-5 is a negative chain of Chr10:17229437-17229291, and BM6-6 is a negative chain of Chr10:17229305-17229173. Understandably, parts of BM6 also include other unlisted DNA fragments located between Chr10:17229173 and 17229587.

[0050] In one specific example, the molecular marker includes at least one region of a subset of BM1, BM2, BM3, BM4, BM5, or BM6.

[0051] In one specific example, the molecular marker includes at least one of the following regions: BM1-1, BM1-2, BM1-3, BM1-4, BM1-5, BM1-6, BM2-1, BM2-2, BM2-3, BM3-1, BM3-2, BM3-3, BM3-4, BM4-1, BM4-2, BM4-3, BM4-4, BM4-5, BM5-1, BM5-2, BM5-3, BM6-1, BM6-2, BM6-3, BM6-4, BM6-4, and BM6-6.

[0052] In a specific example, the molecular markers include at least two of BM1, BM4, and BM6.

[0053] In a specific example, the molecular markers include a combination of partial regions of any two molecular markers among BM1, BM4, and BM6.

[0054] In a specific example, the molecular markers include combinations of any two of the regions BM1-1, BM4-5, and BM6-1. Detecting the methylation levels of any two of these molecular markers has high sensitivity in diagnosing patients with different clinical stages, invasive states, or pathological grades, with no significant decrease in specificity. It has extremely high diagnostic value for early-stage non-muscle-invasive carcinoma.

[0055] In a specific example, the reagent can be used to detect the methylation of molecular markers by one or more of the following methods: methylation-specific PCR, methylation-specific quantitative 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 method, and methylation-sensitive restriction endonuclease method.

[0056] In one specific example, the reagent includes primer pairs and / or probes capable of detecting the methylation level of the aforementioned molecular markers.

[0057] It should be noted that the probe can be a TaqMan probe, labeled with a fluorescent reporter group and a fluorescent quencher group.

[0058] In one specific example, the 5' end of the probe is labeled with a fluorescent reporter group FAM, ROX, or VIC, and the 3' end is labeled with a fluorescent quencher group MGB or BHQ-1. It is understood that the fluorescent groups attached to the probe are not limited to these and can be other fluorescent groups.

[0059] In one specific example, the reagent includes primer pairs for detecting BM1 methylation levels, wherein the primer pairs include at least one of the primer pairs shown in SEQ ID NO. 19–20, SEQ ID NO. 22–23, SEQ ID NO. 25–26, SEQ ID NO. 28–29, SEQ ID NO. 31–32, or SEQ ID NO. 34–35; the reagent includes primer pairs for detecting BM2, wherein the primer pairs include at least one of the primer pairs shown in SEQ ID NO. 37–38, SEQ ID NO. 40–41, or SEQ ID NO. 43–44; and the reagent includes primer pairs for detecting BM3, wherein the primer pairs include the primer pairs shown in SEQ ID NO. 46–47, SEQ ID NO. 49–50, SEQ ID NO. 52–53, or SEQ ID NO. 34–35. The reagent includes at least one of the primer pairs shown in SEQ ID NO. 55-56; the reagent includes a primer pair for detecting BM4, which includes at least one of the primer pairs shown in SEQ ID NO. 58-59, SEQ ID NO. 61-62, SEQ ID NO. 64-65, SEQ ID NO. 67-68, or SEQ ID NO. 70-71; the reagent includes a primer pair for detecting BM5, which includes at least one of the primer pairs shown in SEQ ID NO. 73-74, SEQ ID NO. 76-77, or SEQ ID NO. 79-80; and / or, the reagent includes a primer pair for detecting BM6, which includes the primer pairs shown in SEQ ID NO. 82-83, SEQ ID NO. 85-86, SEQ ID NO. 88-89, SEQ ID NO. 91-92, and SEQ ID NO. 59-80. At least one of the primer pairs shown in NO. 94-95 or SEQ ID NO. 97-98.

[0060] In another specific example, the primer pair is selected from primers that are at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identical to the above sequence.

[0061] In one specific example, the reagent includes a probe for detecting the methylation level of BM1, the probe being at least one of the probes shown in SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 27, SEQ ID NO. 30, SEQ ID NO. 33, or SEQ ID NO. 36; the reagent includes a probe for detecting the methylation level of BM2, the probe being at least one of the probes shown in SEQ ID NO. 39, SEQ ID NO. 42, or SEQ ID NO. 45; the reagent includes a probe for detecting the methylation level of BM3, the probe being at least one of the probes shown in SEQ ID NO. 48, SEQ ID NO. 51, SEQ ID NO. 54, or SEQ ID NO. 57; the reagent includes a probe for detecting the methylation level of BM4, the probe being at least one of the probes shown in SEQ ID NO. 60, SEQ ID NO. 63, SEQ ID NO. 66, SEQ ID NO. 69, or SEQ ID NO. 72; and the reagent includes a probe for detecting the methylation level of BM5, the probe being at least one of the probes shown in SEQ ID NO. 75, SEQ ID NO. 78, or SEQ ID NO. 36. At least one of the probes shown in NO.81; and / or the reagent includes a probe for detecting BM6 methylation levels, the probe including at least one of SEQ ID NO.84, SEQ ID NO.87, SEQ ID NO.90, SEQ ID NO.93, SEQ ID NO.96, or SEQ ID NO.99.

[0062] In another specific example, the probe may be selected from a probe that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identical to the above sequence.

[0063] The application of the above reagents in the preparation of urothelial carcinoma detection products.

[0064] In a specific example, urothelial carcinoma includes bladder cancer and / or upper urinary tract urothelial carcinoma. Optionally, upper urinary tract urothelial carcinoma includes ureteral cancer and renal pelvis cancer.

[0065] Furthermore, one embodiment of this application provides a diagnostic kit for urothelial carcinoma, the kit comprising the reagents described above.

[0066] In a specific example, the diagnosis includes auxiliary diagnosis and / or early diagnosis.

[0067] In a specific example, diagnosing urothelial carcinoma may include diagnosing at least one of bladder cancer, ureteral cancer, and renal pelvis cancer.

[0068] In one specific example, the test kit further includes at least one of a DNA bisulfite conversion reagent, a nucleic acid extraction reagent, a nucleic acid purification reagent, a PCR amplification reagent, and a quality control reagent. Optionally, the test kit also includes a sample collection device.

[0069] In a specific example, the test sample for the test kit can be derived from blood (including whole blood, plasma, and serum), tissue, cells, urine, etc. When the test sample is urine, the test kit can be used for non-invasive testing, reducing patient discomfort during sampling and increasing the kit's availability.

[0070] The diagnostic kit provided in this application is suitable for the diagnosis or auxiliary diagnosis of urothelial carcinomas such as bladder cancer, ureteral cancer, and renal pelvis cancer. It can be used for patients at different clinical stages and has significant diagnostic effects on very early and early-stage cancers. Furthermore, the kit exhibits high sensitivity in diagnosing patients with different pathological grades (including precancerous lesions) and different degrees of invasiveness (including non-invasive cancers). Specific Implementation

[0072] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0073] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0074] Example 1: Establishment of the detection method

[0075] This study used methylation-specific quantitative PCR (qMSP) to detect the methylation levels of six molecular markers in tissue or urine samples from subjects, thereby differentiating urothelial carcinoma patients from healthy individuals. This method requires the design of methylation detection primer pairs and corresponding detection probes for each of the six molecular markers. Using GRCh38.p14 as a reference genome, the DNA sequences of the six molecular markers BM1–BM6, their bisulfite-converted DNA sequences, and their corresponding positions are shown in Table 1.

[0076] Table 1. Location and nucleotide sequence of the target region

[0077]

[0078]

[0079] The sequences shown in SEQ ID NO. 7–18 were artificially synthesized and constructed into the pUC18 plasmid vector, respectively, as methylation and non-methylation templates for qPCR amplification. Multiple pairs of methylation detection primers were designed and artificially synthesized using SEQ ID NO. 7–12 from Table 1 as templates for methylation-specific quantitative PCR reactions. Considering the amplification efficiency of qPCR, the length of each amplicon did not exceed 200 bp. Subsequently, using the plasmids containing the methylated and non-methylated target regions as templates, the performance of each pair of methylation detection primers for each molecular marker was analyzed using the SYBR quantitative PCR system. The amplification curve was required to have a significant exponential growth phase, an amplification efficiency in the range of 95%–105%, and no non-specific amplification. Non-specific amplification refers to the following requirements in a PCR amplification system: when methylated and non-methylated plasmid templates of a molecular marker are added simultaneously, the methylation detection primer pairs should only amplify the methylated plasmid and not the non-methylated plasmid; ② the methylation detection primer pairs should only amplify the target region corresponding to the amplicon and not other non-target regions. After obtaining methylation detection primer pairs that meet the above conditions, corresponding detection probes were designed for each primer pair. All detection probes are TaqMan probes, with a fluorescent group at the 5' end and a fluorescence quencher at the 3' end. It is required that there be no non-specific binding between the detection primer pair and the detection probe, or between the detection probe and the target region. Finally, the effectiveness of the combined use of methylation detection primer pairs and probes was verified in a real-time PCR system. Primer pairs and probes exhibiting exponential amplification were retained as the final detection reagents. Using the above method, six pairs of primers and probes were finally selected to detect BM1 methylation, and their target regions were named BM1-1 to BM1-6, respectively; three pairs of primers and probes were selected to detect BM2 methylation, and their target regions were named BM2-1 to BM2-3, respectively; four pairs of primers and probes were selected to detect BM3 methylation, and their target regions were named BM3-1 to BM3-4, respectively; five pairs of primers and probes were selected to detect BM4 methylation, and their target regions were named BM4-1 to BM4-5, respectively; three pairs of primers and probes were selected to detect BM5 methylation, and their target regions were named BM5-1 to BM5-3, respectively; and six pairs of primers and probes were selected to detect BM6 methylation, and their target regions were named BM6-1 to BM6-6, respectively. The sequences of the methylation detection primer pairs and detection probes used to detect BM1 to BM6 are shown in Table 2. The methylated cytosine sites that can be recognized by each methylation detection primer pair and detection probe are shown in Table 3. The target region amplified by each methylation detection primer pair and its corresponding original DNA sequence are shown in Table 4.

[0080] Table 2. Nucleotide sequences of primer pairs and probes for methylation detection.

[0081]

[0082]

[0083]

[0084] Table 3 shows the methylated cytosine sites that can be identified by the detection primer pairs and probes.

[0085]

[0086]

[0087]

[0088] Table 4. Amplicon and its corresponding unconverted DNA sequence

[0089]

[0090]

[0091] Once the specific methylation detection primer pairs and probes for each molecular marker have been determined, quantitative real-time PCR (qPCR) experiments can be performed to analyze the methylation levels of each target region in the sample. The template for the qPCR reaction is a nucleic acid molecule extracted from the sample and converted to bisulfite; this can be genomic DNA or cell-free DNA. Based on the target region, target-region-specific methylation detection primer pairs and probes are added to the PCR reaction system. If more than one target region is to be detected simultaneously in a single PCR reaction, multiple target region-specific primer pairs and probes must be added. In addition, the detection primer pair and probe for the internal reference gene ACTB must be added to each PCR reaction system to quantify ACTB, thereby monitoring sample quality and interpreting results. The upstream primer sequence for amplifying the ACTB gene fragment used in this invention is 5'-AAGGTGGTTGGGTGGTTGTTTTG-3'SEQ ID NO.154, and the downstream primer sequence is 5'-AATAACACCCCCACCCTGC-3'SEQ ID NO.155. The corresponding detection probe sequence is 5'-GGAGTGGTTTTTGGGTTTG-3'SEQ ID NO.156. The 5' fluorescent group of this detection probe is VIC, and the 3' fluorescent quencher is BHQ1. If only the methylation level of one target region is detected in each PCR system, the 5' fluorescent group of the target region detection probe is FAM, and the 3' fluorescent quencher is MGB. If the methylation level of two or three target regions is detected simultaneously in each PCR reaction system, the 5' fluorescent groups of the detection probes for each target region are different, such as two or three of FAM, ROX, and CY5, while the 3' fluorescent quencher can be the same, such as MGB. The DNA polymerase and buffer used in the PCR reaction system were all purchased from Invitrogen (Cat: 14966005).

[0092] When detecting the methylation level of a target region in a sample, a quality control experiment needs to be set up simultaneously, i.e., a positive control PCR tube and a negative control PCR tube are required. The positive control PCR tube setup is the same as the experimental tubes, but the template is 10... 3 Copy / µL of plasmid containing the transformed target region and 10 3is prepared by mixing equal volumes of plasmids containing the transformed ACTB gene at copies / microliter. Preparation of negative control PCR tubes: the configuration system is the same as that of the experimental tubes, but the template is ultrapure water. After completion of the qPCR reaction, adjust the baseline and set the threshold; the threshold must be located within the exponential amplification phase. The straight line parallel to the X-axis that passes through the threshold is called the threshold line, and the number of cycles corresponding to the intersection of the threshold line and the amplification curve is the Ct value. Analyze the results of the qPCR reaction, which require: ① no amplification in the negative control PCR tube (that is, no amplification curve); ② the positive control PCR tube has an obvious exponential growth phase, and the Ct value of the target gene in the positive control PCR tube is between 26 and 30; ③ the Ct value of the reference gene in the test sample is less than or equal to 33. If the positive control, negative control and reference gene all meet the above requirements, the detection results of the test sample can be analyzed and interpreted. Otherwise, the current experiment is invalid and detection must be re-performed.

[0093] Example 2 Effect of detecting methylation level of target region in tissue samples by qMSP method for diagnosis of bladder cancer

[0094] 1. Sample collection

[0095] Ninety-eight cancer tissue samples from patients with pathologically confirmed bladder cancer and corresponding 98 adjacent tissue samples were collected. All samples were formalin-soaked and paraffin-embedded tissue samples. The collection process of all tissue samples was approved by the Ethics Committee, all volunteers signed informed consent forms, and all tissue samples were processed anonymously.

[0096] 2. Sample DNA extraction

[0097] QIAamp DNA FFPE Tissue Kit (Cat: 56404) was used to extract DNA from tissue samples, and the specific operation was carried out according to the kit instructions.

[0098] 3. Transformation and purification of sample DNA

[0099] Wuhan Amyjet Scientific Co., Ltd. nucleic acid conversion reagent (E Han Medical Preparation No. 20200843) was used to transform and purify sample DNA, and the specific operation steps are shown in the kit instructions.

[0100] 4. qMSP reaction

[0101] Using transformed and purified sample DNA as a template, the detection primer pairs and probes provided in Table 2 were used respectively to perform methylation fluorescence quantitative PCR reaction to amplify each sub-target region. Specifically, after preparing experimental PCR tubes, positive control and negative control PCR tubes according to the method provided in Example 1, prepare the PCR amplification system according to the formula provided in Table 5, and perform PCR amplification according to the program provided in Table 6.

[0102] Table 5 qPCR reaction system

[0103] Platinum II PCR Buffer 5× 5 dNTPs 2.5mM each 3 upstream primers of the target region 10μM 0.5 downstream primers of the target region 10μM 0.5 Target area detection probe 10μM 0.5 ACTB gene upstream primer 10μM 0.5 ACTB gene downstream primer 10μM 0.5 ACTB gene detection probe 10μM 0.5 DNA polymerase / 0.5 DNA of the sample to be tested / 5 Purified water / Add to 25

[0104] Table 6 qPCR reaction procedure

[0105]

[0106] 5. Result Interpretation

[0107] If the internal reference genes in the positive control tube, negative control tube, and experimental tube all meet the quality control requirements in Example 1, the results are interpreted. For tissue samples, if the Ct value in a certain target region is ≤38, the sample is considered methylated positive in that region, and the sample is a positive bladder cancer sample; if the Ct value in a certain target region is >38, the sample is considered methylated negative in that region, and the sample is a negative bladder cancer sample. The sensitivity and specificity of diagnosing bladder cancer tissue samples by using the methylation levels of molecular markers BM1-1 to BM6-6 are shown in Table 7. Sensitivity refers to the proportion of samples with positive histopathological examination that are determined to be positive by this method, and specificity refers to the proportion of samples with negative histopathological examination that are determined to be negative by this method.

[0108] Table 7 Performance of qMSP method in diagnosing bladder cancer tissue samples

[0109]

[0110]

[0111] As shown in Table 7, molecular markers BM1–BM6 exhibit excellent diagnostic performance in bladder cancer tissue samples. Furthermore, for each molecular marker, the diagnostic performance of different regions within its DNA area is similar; for example, there is no significant difference in diagnostic efficacy among BM1-1, BM1-2, BM1-3, BM1-4, BM1-5, and BM1-6. Additionally, molecular markers BM1–BM6 all demonstrate high sensitivity in detecting bladder cancer tissue samples. BM1, BM2, BM4, and BM6 show slightly higher sensitivity than BM3 and BM5, while BM4 and BM6 show slightly higher specificity in detecting adjacent normal tissues than BM1, BM2, BM3, and BM5.

[0112] Example 3: The effect of using the qMSP method to detect the methylation level of a target region in a urine sample and thus diagnose bladder cancer.

[0113] 1. Sample collection

[0114] Urine samples were collected from 140 patients diagnosed with bladder cancer and precancerous lesions by pathological examination, 20 patients diagnosed with ureteral cancer by pathological examination, 35 patients diagnosed with renal pelvis cancer by pathological examination, and 40 healthy individuals undergoing routine physical examinations. In addition, urine samples were collected from 86 patients with common benign urinary tract diseases (including glandular cystitis, urinary tract infection, benign prostatic hyperplasia, kidney stones, hydronephrosis, etc.) and 6 patients with other malignant tumors of the urinary system (renal cancer, prostate cancer, etc.). Each collected urine sample was greater than 50 mL. All sample collection procedures were approved by the ethics committee, all volunteers signed informed consent forms, and all samples were anonymized.

[0115] 2. Extraction of sample DNA

[0116] DNA was extracted from urine samples using a nucleic acid extraction kit from Wuhan Aimeisen Life Science Technology Co., Ltd. (EHanxie Medical Device Registration No. 20210740). The specific operation was performed according to the kit instructions.

[0117] 3. Transformation and purification of sample DNA

[0118] The kits used for DNA transformation and purification in urine samples were all nucleic acid transformation reagents from Wuhan Aimeisen Life Science Technology Co., Ltd. (EHanxie Medical Device Registration No. 20200843). For specific operating procedures, please refer to the kit instructions.

[0119] 4. qMSP reaction

[0120] Methylation-specific quantitative PCR detection was performed according to the method provided in Example 2.

[0121] 5. Result Interpretation

[0122] The difference between the Ct value of the target region and the Ct value of the internal reference gene ACTB in each sample was calculated based on the qPCR results. Then, ROC (receiver-roperating characteristic curve) analysis was performed on all urine samples using IBM SPSS Version 22 software. The sensitivity, specificity, and AUC values ​​at the maximum Youden's index were recorded; this ΔCt value is the cutoff value. If the ΔCt value of the test sample is less than or equal to the cutoff value, the sample is methylated positive in the detected target region, indicating a positive bladder cancer sample. If the ΔCt value of the test sample is greater than the cutoff value, the sample is methylated negative in the detected target region, indicating a negative bladder cancer sample. The performance of the qMSP method in detecting the methylation levels of various molecular markers and diagnosing bladder cancer in urine samples from patients and healthy individuals is shown in Table 8.

[0123] Table 8 Performance of qMSP method in urine samples for diagnosing bladder cancer

[0124]

[0125] From Table 8 and Figures 1-6 It can be seen that molecular markers BM1–BM6 exhibit good performance in diagnosing urine samples from bladder cancer patients, with AUC values ​​greater than 0.78 for all markers. Similar to tissue samples, the AUC values ​​(area under the ROC curve) of different regions within the DNA region of each molecular marker for diagnosing urine samples are not significantly different, indicating that their diagnostic efficacy is essentially consistent. Overall, the sensitivity of molecular markers BM1–BM6 in diagnosing urine samples from bladder cancer patients is lower than that in tissue samples, but their specificity in diagnosing urine samples from healthy individuals is higher than that in detecting adjacent normal tissue samples. Furthermore, the AUC values ​​of molecular markers BM1, BM2, BM4, and BM6 are higher than those of BM3 and BM5, indicating that the diagnostic performance of BM1, BM2, BM4, and BM6 is superior to that of BM3 and BM5. In clinical applications, minimizing false positive results is crucial, thus requiring high specificity from diagnostic reagents and kits. Among the six molecular markers, BM1, BM4, and BM6 demonstrate superior specificity for detecting bladder cancer in urine samples.

[0126] Example 4: The effect of using the qMSP method to detect the methylation level of molecular marker combinations in urine samples for the diagnosis of bladder cancer.

[0127] To further improve the diagnostic performance of molecular markers BM1, BM4, and BM6, this embodiment analyzes the effectiveness of combined diagnosis of urine samples from patients with bladder cancer, other urinary system malignancies, benign urinary system diseases, and healthy individuals. In Examples 4-6, BM1-1, BM4-5, and BM6-1 were selected to represent BM1, BM4, and BM6, respectively.

[0128] The methods for urine sample collection, sample DNA extraction, transformation and purification, and methylation-specific quantitative PCR detection are the same as in Example 3. When two markers are used as target regions, the result interpretation method is as follows: compare the ΔCt value of the sample in the target region with the cutoff value of that region. If the ΔCt value of the sample is less than or equal to the cutoff value, the sample is methylated positive in the target region; if the ΔCt value is greater than the cutoff value, the sample is methylated negative in the target region. If the sample is methylated positive in at least one of the two target regions, the sample is a bladder cancer positive sample; if the sample is methylated negative in both target regions, the sample is a bladder cancer negative sample.

[0129] Based on the above interpretation method, the pathological information of bladder cancer patients was sorted out, and the effectiveness of the BM1+BM6 combination in diagnosing bladder cancer urine samples was statistically analyzed according to different clinical pathological stages, histological grades and pathological types. The results are shown in Tables 9 to 12.

[0130] Table 9. Sensitivity of BM1+BM6 in diagnosing urine samples from bladder cancer patients at different clinical stages.

[0131]

[0132] Table 10 Sensitivity of BM1+BM6 in diagnosing urine samples from bladder cancer patients at different pathological grades

[0133]

[0134] Table 11 Sensitivity of BM1+BM6 in diagnosing urine samples from bladder cancer patients with different invasive states

[0135]

[0136] Table 12 shows the specificity of BM1+BM6 in detecting other urinary system malignancies, benign diseases, and urine samples from healthy individuals.

[0137]

[0138] As shown in Tables 9-12, the combination of molecular markers BM1 and BM6 is highly effective in diagnosing urine samples from patients with bladder cancer, healthy individuals, patients with other malignant tumors of the urinary system, and patients with benign diseases of the urinary system. Specifically, for urine samples from patients with bladder cancer at different pathological stages, the detection rate of the BM1+BM6 combination for precancerous lesions of bladder cancer is 42.9%, and its detection rate for early cancerous changes such as stage 0a and stage 0is is as high as 100%, with an overall diagnostic sensitivity of 96.3%. If urine samples are classified according to different pathological grades of bladder cancer patients, the diagnostic sensitivity of the BM1+BM6 combination is high for all pathological grades, with an overall diagnostic sensitivity of 99.2%. If urine samples are classified according to different invasion states of bladder cancer patients, the detection sensitivity of the BM1+BM6 combination for all samples is close to 100%. In addition, the specificity of the BM1+BM6 combination for detecting samples from healthy individuals and patients with other malignant tumors of the urinary system is 100%, and its specificity for detecting urine samples from patients with benign diseases of the urinary system is 92.7%.

[0139] The pathological information of bladder cancer patients was compiled, and the efficacy of the BM4+BM6 combination in diagnosing bladder cancer urine samples was statistically analyzed according to different clinicopathological stages, histological grades, and pathological types, as shown in Tables 13 to 16.

[0140] Table 13 Sensitivity of BM4+BM6 in diagnosing urine samples from bladder cancer patients at different clinical stages

[0141]

[0142] Table 14 Sensitivity of BM4+BM6 in diagnosing urine samples from bladder cancer patients at different pathological grades

[0143]

[0144] Table 15 Sensitivity of BM4+BM6 in diagnosing urine samples from bladder cancer patients with different invasive states

[0145]

[0146] Table 16 Specificity of BM4+BM6 in detecting other urinary system malignancies, benign diseases, and urine samples from healthy individuals.

[0147]

[0148] As shown in Tables 13-16, the combination of molecular markers BM4 and BM6 is highly effective in diagnosing urine samples from patients with bladder cancer, healthy individuals, patients with other malignant tumors of the urinary system, and patients with benign diseases of the urinary system. For urine samples from bladder cancer patients at different pathological stages, the BM4+BM6 combination showed a detection rate of up to 83.3% for precancerous lesions of bladder cancer, and a 100% detection rate for early cancerous changes such as stage 0a and stage 0is, with an overall diagnostic sensitivity of 94.6%. If urine samples were classified according to different pathological grades of bladder cancer patients, the diagnostic sensitivity of the BM4+BM6 combination for all pathological grades was greater than or equal to 94%, with an overall diagnostic sensitivity of 95.1%. If urine samples were classified according to different invasive states of bladder cancer patients, the sensitivity of the BM4 and BM6 combination in diagnosing invasive cancer (88.9%) was slightly lower than its sensitivity in diagnosing non-invasive cancer (95.8%), with an overall diagnostic sensitivity of 95.1%. In addition, the combination of BM4 and BM6 showed 100% specificity in detecting samples from healthy individuals and patients with other malignant tumors of the urinary system, and 94.9% specificity in detecting urine samples from patients with benign diseases of the urinary system.

[0149] The pathological information of bladder cancer patients was compiled, and the efficacy of the BM1+BM4 combination in diagnosing bladder cancer urine samples was statistically analyzed according to different clinicopathological stages, histological grades, and pathological types, as shown in Tables 17 to 20.

[0150] Table 17 Sensitivity of BM1+BM4 in diagnosing urine samples from bladder cancer patients at different clinical stages

[0151]

[0152]

[0153] Table 18 Sensitivity of BM1+BM4 in diagnosing urine samples from bladder cancer patients at different pathological grades

[0154]

[0155] Table 19 Sensitivity of BM1+BM4 in diagnosing urine samples from bladder cancer patients with different invasive states

[0156]

[0157] Table 20 shows the specificity of BM1+BM4 in detecting other urinary system malignancies, benign diseases, and urine samples from healthy individuals.

[0158]

[0159] As shown in Tables 17-20, the combination of molecular markers BM1 and BM4 demonstrates excellent diagnostic efficacy in urine samples from patients with bladder cancer, healthy individuals, patients with other malignant tumors of the urinary system, and patients with benign diseases of the urinary system. For urine samples from bladder cancer patients at different pathological stages, the detection rate of BM1+BM4 for precancerous lesions of bladder cancer was 71.4%, with detection rates of 90.9% and 100% for early cancerous changes (stages 0a and 0is), respectively. The detection rates for stage I and II bladder cancer patients were both above 93%, with an overall diagnostic sensitivity of 95.4%. When urine samples were classified according to different pathological grades of bladder cancer patients, the diagnostic sensitivity of BM1+BM4 for all pathological grades was greater than 96%, with an overall diagnostic sensitivity of 96.2%. When urine samples were classified according to different invasive states of bladder cancer patients, the sensitivity of BM1+BM4 for diagnosing non-invasive cancer was 97.9%, and the sensitivity for diagnosing invasive cancer was 100%, with an overall diagnostic sensitivity of 98.4%. In addition, the combination of BM1 and BM4 showed 100% specificity in detecting samples from healthy individuals and patients with other malignant tumors of the urinary system, 90.7% specificity in urine samples from patients with benign urinary system diseases, and 93.9% overall specificity in control and interference samples.

[0160] In summary, using any two combinations of biomarkers BM1, BM4, and BM6 as the target region and detecting their methylation levels using the qMSP method can effectively distinguish between bladder cancer patients and non-bladder cancer patients. The diagnostic sensitivity and specificity are both high, and it has a significant diagnostic effect on very early and early-stage bladder cancer.

[0161] Example 5 demonstrates the effectiveness of using the qMSP method to detect the methylation level of molecular marker combinations in urine samples for the diagnosis of ureteral cancer.

[0162] The methods for collecting urine samples, extracting, transforming and purifying sample DNA, and detecting methylation-specific quantitative real-time PCR were the same as in Example 3, and the method for interpreting the results was the same as in Example 4. The sensitivity of the molecular markers BM1+BM6 combination, BM4+BM6 combination, and BM1+BM4 combination in diagnosing urine samples from 20 patients with different pathological stages of ureteral cancer are shown in Tables 21, 22, and 23, respectively.

[0163] Table 21 Sensitivity of BM1+BM6 in diagnosing urine samples from patients with different clinical stages of ureteral cancer

[0164]

[0165]

[0166] Table 22 Sensitivity of BM4+BM6 in diagnosing urine samples from patients with different clinical stages of ureteral cancer

[0167]

[0168] Table 23 Sensitivity of BM1+BM4 in diagnosing urine samples from patients with different clinical stages of ureteral cancer

[0169]

[0170] As shown in Tables 21-23, except for bladder cancer, any combination of two molecular markers BM1, BM4, and BM6 has high detection sensitivity for urine samples from patients with ureteral cancer. The overall detection sensitivity of any combination of two markers for ureteral cancer is greater than or equal to 90%. The detection rates of the BM1+BM6 combination and the BM4+BM6 combination for urine samples from patients with early-stage (stage I and II) ureteral cancer are both 100%, while the detection rates of the BM1+BM4 combination for urine samples from patients with stage I and stage II ureteral cancer are 83.3% and 87.5%, respectively. These results indicate that detecting the methylation level of any combination of two molecular markers BM1, BM4, and BM6 can improve the sensitivity of diagnosing ureteral cancer, especially early-stage patients.

[0171] Example 6 demonstrates the effectiveness of using the qMSP method to detect the methylation level of molecular marker combinations in urine samples for the diagnosis of renal pelvis cancer.

[0172] The methods for collecting urine samples, extracting, transforming and purifying sample DNA, and performing methylation-specific quantitative real-time PCR detection were the same as in Example 3, and the method for interpreting the results was the same as in Example 4. The sensitivities of the molecular markers BM1+BM6 combination, BM4+BM6 combination, and BM1+BM4 combination in diagnosing urine samples from 35 patients with renal pelvis cancer at different pathological stages are shown in Tables 24, 25, and 26, respectively.

[0173] Table 24 Sensitivity of BM1+BM6 in diagnosing urine samples from patients with renal pelvis cancer at different clinical stages

[0174]

[0175] Table 25 Sensitivity of BM4+BM6 in diagnosing urine samples from patients with renal pelvis cancer at different clinical stages

[0176]

[0177] Table 26 Sensitivity of BM1+BM4 in diagnosing urine samples from patients with different clinical stages of renal pelvis cancer.

[0178]

[0179]

[0180] As shown in Tables 24-26, besides bladder cancer and ureteral cancer, any combination of two molecular markers BM1, BM4, and BM6 also exhibits high detection sensitivity in urine samples from patients with renal pelvis cancer. The overall detection sensitivity of any two markers in the combination of renal pelvis cancer is greater than 85%, with the BM4+BM6 combination achieving a total sensitivity of 94.3%. The detection rates of the BM1+BM6 combination and the BM1+BM4 combination in urine samples from patients with early-stage renal pelvis cancer (stage I and II) are both 83.3%, while the detection rates of the BM4+BM6 combination in urine samples from patients with stage I and II renal pelvis cancer are 91.7% and 100%, respectively. These results indicate that detecting the methylation level of any two molecular markers BM1, BM4, and BM6 can improve the sensitivity of diagnosing renal pelvis cancer, especially in early-stage patients.

[0181] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0182] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. The application of a reagent for detecting the methylation level of a molecular marker in the preparation of a urothelial carcinoma detection product, characterized in that, The molecular markers are BM4-1, BM4-2, BM4-3, BM4-4, or BM4-5; Using GRCh38.p14 as the reference genome, BM4-1 is the positive strand of Chr6:27495249-27495413, BM4-2 is the positive strand of Chr6:27495394-27495503, BM4-3 is the positive strand of Chr6:27495524-27495644, BM4-4 is the negative strand of Chr6:27495603-27495461, and BM4-5 is the negative strand of Chr6:27495445-27495376. The reagent includes primer pairs for detecting the methylation levels of BM4-1, BM4-2, BM4-3, BM4-4, or BM4-5. The primer pairs for detecting the methylation level of BM4-1 are the primer pairs shown in SEQ ID NO. 58-59, the primer pairs for detecting the methylation level of BM4-2 are the primer pairs shown in SEQ ID NO. 61-62, the primer pairs for detecting the methylation level of BM4-3 are the primer pairs shown in SEQ ID NO. 64-65, the primer pairs for detecting the methylation level of BM4-4 are the primer pairs shown in SEQ ID NO. 67-68, and the primer pairs for detecting the methylation level of BM4-5 are the primer pairs shown in SEQ ID NO. 70-71. The reagent further includes probes for detecting the methylation levels of BM4-1, BM4-2, BM4-3, BM4-4, or BM4-5. The probe for detecting the methylation level of BM4-1 is SEQ ID NO.60, the probe for detecting the methylation level of BM4-2 is SEQ ID NO.63, the probe for detecting the methylation level of BM4-3 is SEQ ID NO.66, the probe for detecting the methylation level of BM4-4 is SEQ ID NO.69, and the probe for detecting the methylation level of BM4-5 is SEQ ID NO.

72.

2. The application according to claim 1, characterized in that, The molecular markers are either a first combination or a second combination; the first combination is BM4-5 and BM1-1; the second combination is BM4-5 and BM6-1. Using GRCh38.p14 as the reference genome, BM1-1 is a positive strand of Chr10:101140255-101140395; BM6-1 is a positive strand of Chr10:17229173-17229323. The reagents include primer pairs and / or probes for detecting the methylation levels of BM1-1 and / or BM6-1; The reagent includes primer pairs for detecting the methylation level of BM1-1, wherein the primer pairs are those shown in SEQ ID NO.19~20; The reagent includes a primer pair for detecting BM6-1, wherein the primer pair is the primer pair shown in SEQ ID NO. 82~83; The reagent includes a probe for detecting the methylation level of BM1-1, the probe being SEQ ID NO.21; The reagent includes a probe for detecting the methylation level of BM6-1, the probe being SEQ ID NO.

84.

3. The application according to claim 2, characterized in that, The urothelial carcinoma mentioned refers to bladder cancer or upper urinary tract urothelial carcinoma.

4. The application according to claim 3, characterized in that, The upper urinary tract urothelial carcinoma mentioned refers to ureteral carcinoma or renal pelvis carcinoma.

5. A diagnostic kit for urothelial carcinoma, characterized in that, The detection kit includes reagents for detecting the methylation level of molecular markers; The molecular markers are BM4-1, BM4-2, BM4-3, BM4-4, BM4-5, the first combination, the second combination, or the third combination; The first combination is BM4-5 and BM1-1, the second combination is BM4-5 and BM6-1, and the third combination is BM4-5, BM1-1 and BM6-1; The reagents include primer pairs and probes for detecting the molecular markers; The primer pairs for detecting the methylation level of BM4-1 are those shown in SEQ ID NO. 58-59; the primer pairs for detecting the methylation level of BM4-2 are those shown in SEQ ID NO. 61-62; the primer pairs for detecting the methylation level of BM4-3 are those shown in SEQ ID NO. 64-65; the primer pairs for detecting the methylation level of BM4-4 are those shown in SEQ ID NO. 67-68; the primer pairs for detecting the methylation level of BM4-5 are those shown in SEQ ID NO. 70-71; the primer pairs for detecting the methylation level of BM1-1 are those shown in SEQ ID NO. 19-20; and the primer pairs for detecting the methylation level of BM6-1 are those shown in SEQ ID NO. 82-83. The probe for detecting the methylation level of BM4-1 is SEQ ID NO. 60, the probe for detecting the methylation level of BM4-2 is SEQ ID NO. 63, the probe for detecting the methylation level of BM4-3 is SEQ ID NO. 66, the probe for detecting the methylation level of BM4-4 is SEQ ID NO. 69, the probe for detecting the methylation level of BM4-5 is SEQ ID NO. 72, the probe for detecting the methylation level of BM1-1 is SEQ ID NO. 21, and the probe for detecting the methylation level of BM6-1 is SEQ ID NO.

84.

6. The detection kit according to claim 5, characterized in that, The test kit also includes at least one of the following: DNA bisulfite conversion reagent, nucleic acid extraction reagent, nucleic acid purification reagent, PCR amplification reagent, and quality control reagent.

7. The detection kit according to claim 6, characterized in that, The test kit also includes a sample collection device.

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