WO2023142630A1 - Detection method and kit for diagnosing bladder urothelial carcinoma - Google Patents

Abstract

The present invention relates to a detection method and a kit for diagnosing bladder urothelial carcinoma. The present invention provides a use of a primer combination for detecting a DNA methylation difference region in a sample to be tested in preparation of a reagent or a kit for detecting or predicting whether a subject suffers from bladder urothelial carcinoma or whether the type of bladder urothelial carcinoma of a patient is muscle-invasive or non-muscle-invasive, and a kit for detecting or predicting whether a subject suffers from bladder urothelial carcinoma or whether the type of bladder urothelial carcinoma of a patient is muscle-invasive or non-muscle-invasive. The detection method and the kit for diagnosing bladder urothelial carcinoma provided by the present invention have excellent sensitivity and specificity, are of great positive significance in clinical diagnosis, treatment, and later monitoring and prognosis evaluation of bladder urothelial carcinoma, and provide a theoretical basis for further early diagnosis.

Description

A detection method and kit for diagnosis of bladder urothelial carcinoma

Priority and related applications

This application claims the priority of the Chinese patent application submitted to the China Patent Office on January 27, 2022, with the application number 202210097027.1, and the title of the invention is "a detection method and kit for the diagnosis of bladder urothelial carcinoma". The entire contents are incorporated by reference in this application.

technical field

The invention relates to the fields of biotechnology and medical diagnosis, in particular to a detection method and a kit for diagnosing bladder urothelial carcinoma.

Background technique

Bladder cancer (BCa) is the most common malignant tumor of the urinary system, and its incidence is on the rise worldwide. BCa is mainly divided into three pathological types: bladder urothelial carcinoma (Bladder Urothelial Carcinoma, BLCA), squamous cell carcinoma and adenocarcinoma, of which BLCA accounts for 90%, and there will be 212,500 new deaths worldwide in 2020.

BLCA can be further divided into muscle-invasive BCa (muscle-invasive) and non-muscle-invasive BCa (non-muscle-invasive), with non-muscle-invasive BCa accounting for about 75% of all cases. The biological behaviors of the two types of BLCA are different, and the prognosis is very different, so they need to be treated differently. Since non-muscle invasion does not invade the muscular layer of the bladder, urethral bladder tumor resection combined with intracapsular infusion of chemotherapy drugs or BCG can be an effective treatment. Myometrial invasion is more aggressive, grows faster, and has a much higher metastatic potential, usually requiring cystectomy. However, about 70% of clinically diagnosed non-muscle invasive patients do not fully respond to standard practice and eventually relapse or even convert to muscle invasive disease. Delay in surgery due to inaccurate initial pathological diagnosis can increase the hazard ratio for overall death by 50%. Therefore, the early diagnosis of bladder urothelial carcinoma has more important clinical significance.

Currently, the main clinical diagnostic methods for bladder urothelial carcinoma include cystoscopy, urine exfoliation cytology, B-ultrasound, and CT scan. Cystoscopy is regarded as the gold standard for the diagnosis of bladder urothelial carcinoma, but this examination method is invasive and may lead to complications such as urinary tract infection, urethral injury, and bladder injury. Urine exfoliative cytology examinations such as fluorescence in situ hybridization (Fluorescence in situ hybridization, FISH) detection can be used for auxiliary diagnosis of BCa, but the overall sensitivity and specificity are not satisfactory, which are 50% and 85%, respectively. Therefore, it is necessary to develop a new non-invasive bladder urothelial carcinoma detection scheme with higher sensitivity, stronger specificity, and shorter detection cycle. In particular, since bladder urothelial carcinoma has the characteristics of indistinct early symptoms, rapid progression after onset, and difficult pathological diagnosis, it is particularly critical to increase the detection rate and shorten the detection time.

Contents of the invention

The problem to be solved by the invention

The early symptoms of bladder urothelial carcinoma are not obvious, which makes it difficult to detect. Excellent detection methods such as bladder endoscopy are invasive and easily lead to complications. The existing non-invasive detection methods have problems of low sensitivity and specificity. , the present invention provides a detection method and kit for diagnosis of bladder urothelial carcinoma.

solutions to problems

[1]. A primer combination for detecting the DNA methylation difference region in the sample to be tested is prepared for detecting or predicting whether the subject has bladder urothelial carcinoma or whether the patient's type of bladder urothelial carcinoma is The use in a muscle invasive or non-muscle invasive reagent or kit, wherein the differential DNA methylation region is selected from one or more regions in the human hg19 version genome as shown below: chr1: 86038429bp -86548655bp, chr7: 23210805bp-23541085bp, chr12: 50786446bp-51796719bp, chr12: 53396863bp-55397136bp, chr12: 51400340bp-55400617bp, chr5 : 2327659bp-2397867bp, chr5: 5227879bp-5298195bp, chr5: 14341021bp-14541266bp, chr5: 15200260bp-15900461bp , chr8: 2006233bp-2146598bp, chr8: 102136659bp-102436833bp, chr12: 130914602bp-130994836bp, chr17: 77333510bp-77393712bp, chr18: 3869388 bp-3899760bp, chr18: 11741967bp-11762330bp, chr19: 1103012bp-1123479bp and chr3: 180428100bp-1814585400bp.

[2]. The use according to [1], wherein the primer combination includes BLCBSP_V1-4, BLCBSP_V1-9, BLCBSP_V1-13, BLCBSP_V1-14, BLCBSP_V1-15, BLCBSP_V2-7, BLCBSP_V2-8, M N-DMR4-1; Wherein, the forward primer sequence in BLCBSP_V1-4 is shown in SEQ ID NO:1, the reverse primer sequence in BLCBSP_V1-4 is shown in SEQ ID NO:2; The forward primer sequence in BLCBSP_V1-9 is shown in SEQ ID Shown in NO:3, the reverse primer sequence in BLCBSP_V1-9 is shown in SEQ ID NO:4; The forward primer sequence in BLCBSP_V1-13 is shown in SEQ ID NO:5, and the reverse primer sequence in BLCBSP_V1-13 The sequence is shown in SEQ ID NO:6; the forward primer sequence in BLCBSP_V1-14 is shown in SEQ ID NO:7, and the reverse primer sequence in BLCBSP_V1-14 is shown in SEQ ID NO:8; in BLCBSP_V1-15 The forward primer sequence in BLCBSP_V1-15 is shown in SEQ ID NO: 9, the reverse primer sequence in BLCBSP_V1-15 is shown in SEQ ID NO: 10; the forward primer sequence in BLCBSP_V2-7 is shown in SEQ ID NO: 11, The reverse primer sequence among BLCBSP_V2-7 is as shown in SEQ ID NO:12; The forward primer sequence among BLCBSP_V2-8 is as shown in SEQ ID NO:13, and the reverse primer sequence among BLCBSP_V2-8 is as SEQ ID NO: Shown in 14; The forward primer sequence in BLCBSP_V2-10 is shown in SEQ ID NO:15, and the reverse primer sequence in BLCBSP_V2-10 is shown in SEQ ID NO:16; The forward primer sequence in BLCBSP_V2-11 is shown in Shown in SEQ ID NO:17, the reverse primer sequence in BLCBSP_V2-11 is shown in SEQ ID NO:18; The forward primer sequence in BLCBSP_V2-16 is shown in SEQ ID NO:19, and the reverse primer sequence in BLCBSP_V2-16 To primer sequence as shown in SEQ ID NO:20; The forward primer sequence among BLCBSP_V2-17 is as shown in SEQ ID NO:21, and the reverse primer sequence among BLCBSP_V2-17 is as shown in SEQ ID NO:22; BLCBSP_V2- The forward primer sequence in 18 is shown in SEQ ID NO:23, the reverse primer sequence in BLCBSP_V2-18 is shown in SEQ ID NO:24; The forward primer sequence in BLCBSP_V2-19 is shown in SEQ ID NO:25 Shown, the reverse primer sequence in BLCBSP_V2-19 is shown in SEQ ID NO:26; The forward primer sequence in BLCBSP_V2-21 is shown in SEQ ID NO:27, and the reverse primer sequence in BLCBSP_V2-21 is shown in SEQ ID Shown in NO:28; The forward primer sequence among BLCBSP_V2-22 is shown in SEQ ID NO:29, and the reverse primer sequence among BLCBSP_V2-22 is shown in SEQ ID NO:30; The forward primer among BLCBSP_V2-24 The sequence is shown in SEQ ID NO:31, the reverse primer sequence in BLCBSP_V2-24 is shown in SEQ ID NO:32; the forward primer sequence in BLCBSP_V2-33 is shown in SEQ ID NO:33, in BLCBSP_V2-33 The reverse primer sequence in BLCBSP_V2-35 is shown in SEQ ID NO:34; The forward primer sequence in BLCBSP_V2-35 is shown in SEQ ID NO:35, and the reverse primer sequence in BLCBSP_V2-35 is shown in SEQ ID NO:36; The forward primer sequence in MN-DMR4-1 is shown in SEQ ID NO:37, and the reverse primer sequence in MN-DMR4-1 is shown in SEQ ID NO:38.

[3]. The use according to [1] or [2], wherein the sample to be tested is urine cfDNA or bladder urothelial carcinoma tissue gDNA derived from the subject.

[4]. A kit for detecting or predicting whether a subject suffers from bladder urothelial carcinoma or whether the type of bladder urothelial carcinoma is muscle-invasive or non-muscle-invasive, which comprises A multiplex PCR reagent for detecting DNA methylation differential regions in samples to be tested, wherein the DNA methylation differential regions are selected from one or more regions in the human hg19 version genome as shown below: chr1: 86038429bp- 86548655bp, chr7: 23210805bp-23541085bp, chr12: 50786446bp-51796719bp, chr12: 53396863bp-55397136bp, chr12: 51400340bp-55400617bp, chr5 : 2327659bp-2397867bp, chr5: 5227879bp-5298195bp, chr5: 14341021bp-14541266bp, chr5: 15200260bp-15900461bp, chr8: 2006233bp-2146598bp, chr8: 102136659bp-102436833bp, chr12: 130914602bp-130994836bp, chr17: 77333510bp-77393712bp, chr18: 3869388b p-3899760bp, chr18: 11741967bp-11762330bp, chr19: 1103012bp-1123479bp and chr3: 180428100bp-1814585400bp.

[5]. The kit according to [4], wherein the multiplex PCR reagents include a primer set, a positive reference and a negative reference.

[6]. The kit according to [5], wherein the primer combination includes BLCBSP_V1-4, BLCBSP_V1-9, BLCBSP_V1-13, BLCBSP_V1-14, BLCBSP_V1-15, BLCBSP_V2-7, BLCBSP_V2-8 , BLCBSP_V2-10, BLCBSP_V2-11, BLCBSP_V2-16, BLCBSP_V2-17, BLCBSP_V2-18, BLCBSP_V2-19, BLCBSP_V2-21, BLCBSP_V2-22, BLCBSP_V2-24, BLCBSP_V2-33, BLCBSP_V2-35 , MN-DMR4-1 ; Wherein, the forward primer sequence among BLCBSP_V1-4 is as shown in SEQ ID NO:1, and the reverse primer sequence among BLCBSP_V1-4 is as shown in SEQ ID NO:2; The forward primer sequence among BLCBSP_V1-9 is as shown in SEQ ID NO:2; Shown in ID NO:3, the reverse primer sequence in BLCBSP_V1-9 is shown in SEQ ID NO:4; The forward primer sequence in BLCBSP_V1-13 is shown in SEQ ID NO:5, and the reverse in BLCBSP_V1-13 The primer sequence is shown in SEQ ID NO:6; the forward primer sequence in BLCBSP_V1-14 is shown in SEQ ID NO:7, and the reverse primer sequence in BLCBSP_V1-14 is shown in SEQ ID NO:8; BLCBSP_V1-15 The forward primer sequence in BLCBSP_V1-15 is shown in SEQ ID NO:10; the forward primer sequence in BLCBSP_V2-7 is shown in SEQ ID NO:11 , the reverse primer sequence in BLCBSP_V2-7 is shown in SEQ ID NO:12; The forward primer sequence in BLCBSP_V2-8 is shown in SEQ ID NO:13, and the reverse primer sequence in BLCBSP_V2-8 is shown in SEQ ID NO Shown in: 14; The forward primer sequence in BLCBSP_V2-10 is shown in SEQ ID NO:15, and the reverse primer sequence in BLCBSP_V2-10 is shown in SEQ ID NO:16; The forward primer sequence in BLCBSP_V2-11 As shown in SEQ ID NO: 17, the reverse primer sequence in BLCBSP_V2-11 is shown in SEQ ID NO: 18; The forward primer sequence in BLCBSP_V2-16 is shown in SEQ ID NO: 19, and the sequence in BLCBSP_V2-16 The reverse primer sequence is shown in SEQ ID NO:20; the forward primer sequence in BLCBSP_V2-17 is shown in SEQ ID NO:21, and the reverse primer sequence in BLCBSP_V2-17 is shown in SEQ ID NO:22; BLCBSP_V2 The forward primer sequence in -18 is shown in SEQ ID NO:23, the reverse primer sequence in BLCBSP_V2-18 is shown in SEQ ID NO:24; The forward primer sequence in BLCBSP_V2-19 is shown in SEQ ID NO:25 Shown, the reverse primer sequence in BLCBSP_V2-19 is shown in SEQ ID NO:26; The forward primer sequence in BLCBSP_V2-21 is shown in SEQ ID NO:27, and the reverse primer sequence in BLCBSP_V2-21 is shown in SEQ ID NO:27 Shown in ID NO:28; The forward primer sequence in BLCBSP_V2-22 is shown in SEQ ID NO:29, and the reverse primer sequence in BLCBSP_V2-22 is shown in SEQ ID NO:30; The forward primer sequence in BLCBSP_V2-24 The primer sequence is shown in SEQ ID NO:31, the reverse primer sequence in BLCBSP_V2-24 is shown in SEQ ID NO:32; The forward primer sequence in BLCBSP_V2-33 is shown in SEQ ID NO:33, BLCBSP_V2-33 The reverse primer sequence in BLCBSP_V2-35 is shown in SEQ ID NO:34; The forward primer sequence in BLCBSP_V2-35 is shown in SEQ ID NO:35, and the reverse primer sequence in BLCBSP_V2-35 is shown in SEQ ID NO:36 The forward primer sequence in MN-DMR4-1 is shown in SEQ ID NO:37, and the reverse primer sequence in MN-DMR4-1 is shown in SEQ ID NO:38.

[7]. The kit according to any one of [4] to [6], wherein the sample to be tested is urine cfDNA or bladder urothelial carcinoma tissue derived from the subject gDNA.

[8]. A method for detecting or predicting whether a subject suffers from bladder urothelial carcinoma or whether the type of bladder urothelial carcinoma is muscle-invasive or non-muscle-invasive, which includes the following steps: extracting The urine cfDNA of the subjects was converted to bisulfite, and then multiplex PCR was performed with primer combinations and the products were recovered. Index PCR and high-throughput sequencing were performed on the multiplex PCR products to calculate the muscle-invasive urothelial carcinoma of the bladder, The methylation level of the DNA methylation difference region between non-muscle-invasive urothelial carcinoma of the bladder and normal bladder tissue, to determine whether the subject has bladder urothelial carcinoma or whether the patient type of bladder urothelial carcinoma is Is muscle invasive or non-muscle invasive; where,

The primer combination includes BLCBSP_V1-4, BLCBSP_V1-9, BLCBSP_V1-13, BLCBSP_V1-14, BLCBSP_V1-15, BLCBSP_V2-7, BLCBSP_V2-8, BLCBSP_V2-10, BLCBSP_V2-11, BLCBSP_V2-16, BLCBSP_V2-17 、BLCBSP_V2 -18, BLCBSP_V2-19, BLCBSP_V2-21, BLCBSP_V2-22, BLCBSP_V2-24, BLCBSP_V2-33, BLCBSP_V2-35, MN-DMR4-1; wherein, the forward primer sequence in BLCBSP_V1-4 is as SEQ ID NO:1 Shown, the reverse primer sequence in BLCBSP_V1-4 is shown in SEQ ID NO:2; The forward primer sequence in BLCBSP_V1-9 is shown in SEQ ID NO:3, and the reverse primer sequence in BLCBSP_V1-9 is shown in SEQ ID NO: Shown in ID NO:4; The forward primer sequence in BLCBSP_V1-13 is shown in SEQ ID NO:5, and the reverse primer sequence in BLCBSP_V1-13 is shown in SEQ ID NO:6; The forward primer sequence in BLCBSP_V1-14 The primer sequence is shown in SEQ ID NO:7, the reverse primer sequence in BLCBSP_V1-14 is shown in SEQ ID NO:8; the forward primer sequence in BLCBSP_V1-15 is shown in SEQ ID NO:9, BLCBSP_V1-15 The reverse primer sequence in BLCBSP_V2-7 is shown in SEQ ID NO:10; the forward primer sequence in BLCBSP_V2-7 is shown in SEQ ID NO:11, and the reverse primer sequence in BLCBSP_V2-7 is shown in SEQ ID NO:12 ; The forward primer sequence in BLCBSP_V2-8 is shown in SEQ ID NO:13, and the reverse primer sequence in BLCBSP_V2-8 is shown in SEQ ID NO:14; The forward primer sequence in BLCBSP_V2-10 is shown in SEQ ID NO Shown in: 15, the reverse primer sequence in BLCBSP_V2-10 is shown in SEQ ID NO: 16; The forward primer sequence in BLCBSP_V2-11 is shown in SEQ ID NO: 17, the reverse primer sequence in BLCBSP_V2-11 As shown in SEQ ID NO:18; The forward primer sequence among BLCBSP_V2-16 is as shown in SEQ ID NO:19, and the reverse primer sequence among BLCBSP_V2-16 is as shown in SEQ ID NO:20; Among the BLCBSP_V2-17 The forward primer sequence is shown in SEQ ID NO:21, the reverse primer sequence in BLCBSP_V2-17 is shown in SEQ ID NO:22; the forward primer sequence in BLCBSP_V2-18 is shown in SEQ ID NO:23, BLCBSP_V2 The reverse primer sequence in -18 is shown in SEQ ID NO:24; The forward primer sequence in BLCBSP_V2-19 is shown in SEQ ID NO:25, and the reverse primer sequence in BLCBSP_V2-19 is shown in SEQ ID NO:26 Shown; The forward primer sequence in BLCBSP_V2-21 is shown in SEQ ID NO:27, and the reverse primer sequence in BLCBSP_V2-21 is shown in SEQ ID NO:28; The forward primer sequence in BLCBSP_V2-22 is shown in SEQ ID NO:28 Shown in ID NO:29, the reverse primer sequence in BLCBSP_V2-22 is shown in SEQ ID NO:30; The forward primer sequence in BLCBSP_V2-24 is shown in SEQ ID NO:31, the reverse in BLCBSP_V2-24 The primer sequence is shown in SEQ ID NO:32; The forward primer sequence in BLCBSP_V2-33 is shown in SEQ ID NO:33, and the reverse primer sequence in BLCBSP_V2-33 is shown in SEQ ID NO:34; BLCBSP_V2-35 The forward primer sequence in BLCBSP_V2-35 is shown in SEQ ID NO: 36; the forward primer sequence in MN-DMR4-1 is shown in SEQ ID NO: 37 Shown, the reverse primer sequence in MN-DMR4-1 is shown in SEQ ID NO:38;

The DNA methylation differential region is selected from one or more regions in the human hg19 version genome as follows: chr1: 86038429bp-86548655bp, chr7: 23210805bp-23541085bp, chr12: 50786446bp-51796719bp, chr12: 53396863bp-5 5397136bp, chr12: 51400340bp-55400617bp, chr5: 2327659bp-2397867bp, chr5: 5227879bp-5298195bp, chr5: 14341021bp-14541266bp, chr5: 15200260bp-15900 461bp, chr8: 2006233bp-2146598bp, chr8: 102136659bp-102436833bp, chr12: 130914602bp-130994836bp, chr17: 77333510bp-77393712bp, chr18: 3869388bp-3899760bp, chr18: 11741967bp-11762330bp, chr19: 1103012bp-1123479bp and chr3: 180428100bp-181458 5400bp.

The effect of the invention

Through the implementation of the above technical solution, the detection method and kit for the diagnosis of bladder urothelial carcinoma provided by the present invention have excellent sensitivity and specificity. The experimental data show that when using urine to diagnose whether a subject has bladder urothelial carcinoma, the sensitivity is 0.889 and the specificity is 0.945; For invasive bladder urothelial carcinoma, the sensitivity was 0.850 and the specificity was 1.000. It is of great positive significance to the clinical diagnosis, treatment, and later monitoring and prognosis evaluation of bladder urothelial carcinoma, and provides a theoretical basis for further early diagnosis.

Description of drawings

Figure 1 is the methylation level curve of the model standard.

Figure 2 is the tumor DNA detection results of bladder urothelial cancer cell lines RT4 and 5637 biological standards.

Figure 3 shows the detection results of tumor DNA in tissues and urine of 121 real samples.

Detailed ways

Embodiments of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the configurations described below, and various changes can be made within the scope of the claims for protection of the invention. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in this document. within the technical scope of the invention.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present invention, the term "one (a)" or "one (an)" or "one (the)" may refer to "one", and may also refer to "one or more", "at least one" and "one or more than one".

In the present invention, the words "comprising", "having", "including" or "containing" mean inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

As used herein, the term "about" means that a value includes the standard deviation of error of the apparatus or method used to determine the value.

Although the disclosure supports the definition of the term "or" as an alternative only and "and/or", the term "or" in this disclosure means "and / or".

In the present invention, unless otherwise stated, the position numbers of human chromosomes in this disclosure are all numbered based on the hg19 version.

In the present invention, the principle of DNA bisulfite conversion is to treat the sample to be tested with bisulfite, convert the unmethylated C base in the genome into U, and turn it into T after PCR amplification, and The C bases that originally had methylation modifications were distinguished, combined with high-throughput sequencing technology, and compared with the reference sequence, it was possible to determine whether methylation occurred at the CpG/CHG/CHH site.

In the present invention, "cfDNA" refers to free DNA (Cell-Free Circulating DNA), which is the partly degraded endogenous DNA in the body that is free from extracellular and exists in human circulating blood, urine and other body fluids. The length of cfDNA is mostly below 200bp.

In the present invention, "gDNA" refers to "genome DNA", which refers to the total DNA content of an organism in a haploid state.

In the present invention, "DNA methylation" refers to transfer of methyl group to specific base process. In mammals, methylation is predominantly at the 5'C-terminus of nucleotide cytosine residues. In the human genome, a large amount of DNA methylation occurs on cytosine in CpG dinucleotides, C is cytosine, G is guanine, and p is a phosphate group. DNA methylation can also occur in cytosine in nucleotide sequences such as CHG and CHH, where H is adenine, cytosine or thymine. DNA methylation can also occur on non-cytosines, such as N6-methyladenine. In addition, DNA methylation can also be in the form of 5-hydroxymethylcytosine.

In the present invention, "DNA methylation difference region", "DNA differential methylation region" and "DNA methylation modification difference region" can be used interchangeably, all refer to the degree of DNA methylation modification in two groups of biological samples A genomic region consisting of multiple adjacent base positions that differ in .

In this disclosure, "DNA methylation modification" can be expressed as a single base methylation modification, or as the average value of multiple base methylation modifications on a continuous region, or as is a methylated haplotype, and can also be expressed as a methylated haplotype.

In this disclosure, "DNA methylation modification degree", "DNA methylation modification level" and "methylation level" are used interchangeably, which can be used as an overall quantitative parameter β or the abundance of methylated haplotypes. degree to describe.

In the present disclosure, the overall quantitative parameter β of DNA methylation modification level can be expressed as the average degree of methylation modification of C bases, that is: β=number of C carrying methylation modification/(carrying methylation The number of modified Cs + the number of Cs not carrying methylated modifications).

In the present invention, "methylation haplotype" is also referred to as "methylation haplotype", which refers to the methylation modification of two or more consecutive C base sites on a single DNA fragment The combination. Since the DNA methylation modification is correlated on the same chromosome (DNA chain), there may be multiple adjacent C base sites, and their methylation modification conditions are related to each other to form a certain fixed form. Therefore, DNA methylation haplotype is a linkage pattern in which DNA methylation modification exists on the same chromosome.

In the present invention, the "abundance of methylated haplotypes" refers to the proportion of specific methylated haplotypes obtained after statistical analysis of methylated haplotype information. That is: the abundance of methylated haplotype = the number of DNA fragments corresponding to a specific methylated haplotype/the total number of DNA fragments completely covering the genomic region of the methylated haplotype.

In some specific embodiments, the present invention provides regions with significant differences in DNA methylation between patients with bladder urothelial carcinoma and normal individuals, and DNA differences between muscle-invasive and non-muscle-invasive bladder urothelial carcinoma Methylation region, specifically, the DNA methylation differential region is selected from one or more regions in the human hg19 version genome as follows: chr1: 86038429bp-86548655bp, chr7: 23210805bp-23541085bp, chr12: 50786446bp -51796719bp, chr12: 53396863bp-55397136bp, chr12: 51400340bp-55400617bp, chr5: 2327659bp-2397867bp, chr5: 5227879bp-5298195bp, chr5: 1434 1021bp-14541266bp, chr5: 15200260bp-15900461bp, chr8: 2006233bp-2146598bp, chr8: 102136659bp-102436833bp , chr12: 130914602bp-130994836bp, chr17: 77333510bp-77393712bp, chr18: 3869388bp-3899760bp, chr18: 11741967bp-11762330bp, chr19: 1103012 bp-1123479bp and chr3: 180428100bp-1814585400bp.

In the present invention, "multiple PCR" is also called multiple primer PCR or composite PCR. It is a PCR reaction in which more than two pairs of primers are added to the same PCR reaction system to simultaneously amplify multiple nucleic acid fragments. The reaction principle, reaction Reagents and operating procedures are the same as general PCR.

In some specific embodiments, the present invention provides DNA methylation regions with obvious differences between patients with bladder urothelial carcinoma and normal people and DNA between muscle-invasive and non-muscle-invasive bladder urothelial carcinoma. Multiplex PCR primer sets designed for differentially methylated regions.

In some preferred embodiments, the multiplex PCR primer combination provided by the present invention includes BLCBSP_V1-4, BLCBSP_V1-9, BLCBSP_V1-13, BLCBSP_V1-14, BLCBSP_V1-15, BLCBSP_V2-7, BLCBSP_V2-8, BLCBSP_V2-10, BLCBSP_V2 -11, BLCBSP_V2-16, BLCBSP_V2-17, BLCBSP_V2-18, BLCBSP_V2-19, BLCBSP_V2-21, BLCBSP_V2-22, BLCBSP_V2-24, BLCBSP_V2-33, BLCBSP_V2-35, MN-DMR4-1; among them, BLCBSP_V1- 4 The forward primer sequence in BLCBSP_V1-4 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNCTTAAAAAATATCTCCCCCATCT-3' (SEQ ID NO: 2) ); BLCBSP_V1-9 The forward primer sequence in BLCBSP_V1-9 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCTCATTTATCCTAAAACCTTTTC-3' (SEQ ID NO: 4). ); BLCBSP_V1-13 The forward primer sequence in BLCBSP_V1-13 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNNTCTATTACTAAAACCCCAAAA-3' (SEQ ID NO: 6); BLCBSP_V1-14 The forward primer sequence in BLCBSP_V1-14 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNNCAAAACTATTCTACCATAACAAAC-3' (SEQ ID NO:8); BLCBSP_V1-15 The forward primer sequence in BLCBSP_V1-15 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNNNNTGAGGGATTTTTAAAAGGGG-3'(SEQ ID NO:9), the reverse primer sequence in BLCBSP_V1-15 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNACCCTACCAAAAAAA CTATTCTT-3'(SEQ ID NO:10); BLCBSP_V2- The forward primer sequence in 7 is 5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGGTTTAGTTTTTAGATATTAGAG-3' (SEQ ID NO: 11), and the reverse primer sequence in BLCBSP_V2-7 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNAAACTAACCCTATAACCCCA-3' (SEQ ID NO :12); BLCBSP_V2- The forward primer sequence in 8 is 5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNTTTTATATAAAATTTTTGTGGATGG-3' (SEQ ID NO: 13), and the reverse primer sequence in BLCBSP_V2-8 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNACCTAAATACTTTAAACTAACCT-3' (SEQ ID NO:14); BLCBSP_V2- The forward primer sequence in 10 is 5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGTTTGGGTATTTTATTTGTGAAA-3' (SEQ ID NO:15), and the reverse primer sequence in BLCBSP_V2-10 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCAAAAACAAACACACAAACTAAC-3' (SEQ ID NO:16); BLCBSP_V2- The forward primer sequence in 11 is 5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGGGGATTYGTAATAAGTGG-3' (SEQ ID NO: 17), and the reverse primer sequence in BLCBSP_V2-11 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCRAACAAACCCCTAAACTC-3' (SEQ ID NO:18); BLCBSP_V2- The forward primer sequence in 16 is 5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGTGTGGTTTGAGTGTTTGTT-3' (SEQ ID NO:19), and the reverse primer sequence in BLCBSP_V2-16 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNAATCCTTCACTCAATCCCAC-3' (SEQ ID NO:20); BLCBSP_V2- The forward primer sequence in 17 is 5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNGGAAATTATTTTGGGGATTGAG-3' (SEQ ID NO:21), and the reverse primer sequence in BLCBSP_V2-17 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNTAAACCAAATATTTCAAAAAAACC-3' (SE Q ID NO:22); BLCBSP_V2- The forward primer sequence in 18 is 5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNAGGAAGGGGTTTTAGTTAAAG-3' (SEQ ID NO: 23), and the reverse primer sequence in BLCBSP_V2-18 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNCCTAAAACAAAAATCAAAATATCC-3' (SEQ ID NO:24); BLCBSP_V2- The forward primer sequence in 19 is 5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNGGTAGGAGAAGGTTATTTATG-3' (SEQ ID NO:25), and the reverse primer sequence in BLCBSP_V2-19 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCTTTTTAAAATATAACACCCATCC-3' (SEQ ID NO:26); BLCBSP_V2- The forward primer sequence in 21 is 5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGAGGAATTTTTTTTTGGTAGGA-3' (SEQ ID NO:27), and the reverse primer sequence in BLCBSP_V2-21 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCCCTAATTACAATACTAACACT-3' (SEQ ID NO:28); BLCBSP_V2- The forward primer sequence in 22 is 5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNGATTTGGTGAAGAGTTTTTGG-3' (SEQ ID NO:29), and the reverse primer sequence in BLCBSP_V2-22 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNTAACTACACACCCTACAATAC-3' (SEQ ID NO:30); BLCBSP_V2- The forward primer sequence in 24 is 5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGTTGAGYGAGGTTTGGAG-3' (SEQ ID NO: 31), and the reverse primer sequence in BLCBSP_V2-24 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNAAATCTACTAAATAAATACAACACA-3' (SEQ ID NO:32); BLCBSP_V2- The forward primer sequence in 33 is 5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNTGTTGGGAAAGGGAGGG-3' (SEQ ID NO:33), and the reverse primer sequence in BLCBSP_V2-33 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNCCCAACTTCACTTTTTCACT-3' (SEQ ID NO:34); BLCBSP_V2- The forward primer sequence in 35 is 5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNTGGGGTTTAGAGGGATGG-3' (SEQ ID NO:35), and the reverse primer sequence in BLCBSP_V2-35 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNTCTAATAACCCCTACTAATCAC-3' (SEQ ID NO:36); MN- The forward primer sequence in DMR4-1 is 5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGTTAGGGTTTGTAGGGTTT-3' (SEQ ID NO:37), and the reverse primer sequence in MN-DMR4-1 is 5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNTAATAAAACTAAACTACACTAACCTC-3' (SEQ ID NO:38 ).

In order to express the technical solution of the present invention more clearly, the following will be further described in conjunction with specific examples, but they cannot be used to limit the present invention, which are only some examples of the present invention. Unless otherwise stated, the instruments, reagents, materials, experimental animals, etc. used in the present invention can be obtained through conventional commercial means.

Example 1

The present invention is based on performing single-cell transcriptome, single-cell chromatin accessibility sequencing and Whole-genome DNA methylation sequencing, analysis found that cells of different origin types have different DNA methylation characteristics, causing differences in the degree of chromatin opening, which in turn affects differences in gene expression, and ultimately leads to different fates of cells. Some are normal cells, while some develop into muscle-invasive cancer cells, or non-muscle-invasive cancer cells. Through the statistical analysis of these DNA methylation features, the regions (DMRs) with significant differences in methylation levels in the three source cells were screened out, as shown in Table 1. The present invention is based on the method of methylation multiple PCR, amplifies these DMR regions, and detects the methylation level on these DNA regions by high-throughput sequencing, and the specific primer combinations are shown in Table 2:

Table 1 Differentially methylated regions for detection of bladder urothelial carcinoma

DMR ID NO.	chromosome number	physical location	Genome version number
1	1	86038429-86548655	hg19
2	7	23210805-23541085	hg19
3	12	50786446-51796719	hg19
4	12	53396863-55397136	hg19
5	12	51400340-55400617	hg19
6	5	2327659-2397867	hg19
7	5	5227879-5298195	hg19
8	5	14341021-14541266	hg19
9	5	15200260-15900461	hg19
10	8	2006233-2146598	hg19
11	8	102136659-102436833	hg19
12	12	130914602-130994836	hg19
13	17	77333510-77393712	hg19
14	18	3869388-3899760	hg19
15	18	11741967-11762330	hg19
16	19	1103012-1123479	hg19
17	3	180428100-1814585400	hg19

Table 2 Primer combinations for methylation multiplex PCR

All primers have adapter universal sequence and 7-base unimolecular marker sequence:

Forward primer adapter universal sequence:

5'-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNN,

General sequence of reverse primer adapter:

5'-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNN.

The primers used in the present invention were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

Example 2

17 methylated regions were detected using commercial fully methylated and unmethylated pattern standards (purchased from Zymo).

The specific process is as follows:

1. DNA bisulfite conversion (BS treatment)

The DNA bisulfite conversion kit was purchased from Zymo, and the conversion product was eluted with NF-H ₂ O according to the instructions of the kit.

2. Model Standard Gradient Incorporation

Single-strand quantification was performed after BS-treated fully methylated and unmethylated standard samples were disrupted by ultrasound. Carry out gradient to incorporate according to DNA concentration, the application has set up 13 pattern standard substance gradients altogether (methylation level is 100%, 99%, 98%, 95%, 90%, 80%, 50%, 20%, 10% %, 5%, 2%, 1%, 0%).

3. Preparation of Methylated Multiplex PCR Primer Sets

19 pairs of primers were mixed for later use.

4. PCR master mix configuration

Prepare the premix according to Table 3. Reagents for multiplex PCR reactions were purchased from Thermo Fisher (N8080241).

Table 3 Methylation multiplex PCR master mix configuration scheme

Reagent	Volume (μL)
DNA (20ng initial amount)	x
10×Buffer	2
10mM dNTPs	0.4
25mM MgCl2	1.2
Amplitaq Goldtaq	0.1
Primer	4.6
NF-H 2 O	11.7‐X
Total	20

PCR reaction program: 10min at 95°C; 25×(30s at 95°C, 30s at 65°C, 2min at 54°C, 30s at 65°C, 30s at 72°C); 10min at 72°C; store at 16°C.

The reaction product can be temporarily stored at -20°C.

5. Multiplex PCR product recovery

1) Pipette the product in the PCR tube into the corresponding 1.5mL centrifuge tube with 24μL of XP Beads added.

2) Vortex to mix for 15 seconds, centrifuge for 3 seconds, and incubate at room temperature for 5 minutes.

3) After the incubation is completed, place the 1.5mL centrifuge tube on the magnetic stand, let it stand until the liquid is clear (about 5min), and discard the supernatant.

4) Add 180 μL of freshly prepared 80% ethanol, invert up and down, left and right sides twice, incubate on a magnetic stand at room temperature for 30 seconds, and discard the supernatant.

5) Repeat step 4) once.

6) Remove the 1.5mL centrifuge tube from the magnetic stand and centrifuge for 30s. Then put the 1.5mL centrifuge tube back into the magnetic stand, let it stand for 1min, and use a 20μL pipette to discard the residual liquid.

7) Open the cap of the 1.5mL centrifuge tube and dry it at room temperature until the surface of the magnetic beads becomes dull (about 1-3min).

8) Remove the 1.5 mL centrifuge tube from the magnetic stand, and add 25 μL NF-H ₂ O. After flicking with your fingers, vortex and shake to mix for 5 seconds, and then incubate at room temperature for 5 minutes.

9) After the incubation is complete, place the 1.5mL centrifuge tube on the magnetic stand, let it stand until the liquid is clear, and set aside.

6. index PCR

Reagents for index PCR were purchased from KAPA Biosystems (KK2602).

The reaction system is shown in Table 4:

Table 4 index PCR reaction system

Reagent	Volume (μL)
Recycled product from previous step	twenty three
2×PCR mix	25
i5 index	1
i7 index	1
Total	50

PCR reaction program: 98°C for 45s; 13× (98°C for 15s, 60°C for 30s, 72°C for 30s); 72°C for 5min; 4°C for storage.

The reaction product can be placed at -20°C and recovered the next day.

7. Index PCR product recovery and quantification

1) Pipette the product in the PCR tube into the corresponding 1.5mL centrifuge tube with 60μL of XP Beads added.

2) Vortex to mix for 15 seconds, centrifuge for 3 seconds, and incubate at room temperature for 5 minutes.

3) After the incubation is completed, place the 1.5mL centrifuge tube on the magnetic stand, let it stand until the liquid is clear (about 5min), and discard the supernatant.

4) Add 180 μL of freshly prepared 80% ethanol, invert up and down, left and right twice, incubate on a magnetic stand at room temperature for 30 seconds, and discard the supernatant.

5) Repeat step 4) once.

6) Remove the 1.5mL centrifuge tube from the magnetic stand and centrifuge for 30s. Then put the 1.5mL centrifuge tube back into the magnetic stand, let it stand for 1min, and use a 20μL pipette to discard the residual liquid.

7) Open the cap of the 1.5mL centrifuge tube and dry it at room temperature until the surface of the magnetic beads becomes dull (about 1-3min).

8) Remove the 1.5mL centrifuge tube from the magnetic stand and add 36μL EB. After flicking with a finger, Vortex was mixed for 5 seconds, and then incubated at room temperature for 5 minutes.

9) After the incubation is complete, place the 1.5mL centrifuge tube on the magnetic stand, let it stand until the liquid is clear (about 5min), and transfer to a new 1.5mL centrifuge tube.

10) Double-strand quantification using dsDNA Qubit.

8. Library on computer

After the concentration of the library was determined by fluorescent quantitative PCR and qualified, the Illumina Novaseq platform was used for PE150 sequencing, and the data volume of each library was 5-8M reads.

9. Results

The detected 13 pattern standards with different gradients have good discrimination of methylation level, no matter in the DNA template of high methylation or low methylation, most of the sites have no methylation difference of 1%. Can carry out very good identification, and the repeatability of 3 times is good. Figure 1 is the methylation level curves of 13 different gradient mode standards after amplification by 19 pairs of primers (17 regions).

Example 3

Use bladder urothelial cancer cell lines RT4 and 5637 to detect biological standards to clarify the minimum detection limit of this application for bladder urothelial cancer and its typing

The specific process is as follows:

1. gDNA extraction

Bladder urothelial carcinoma cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences. The extraction kit was purchased from QIAGEN (69506), and was carried out according to the instructions of the kit.

2. DNA bisulfite conversion (reference example 2)

3. Biological Standard Gradient Incorporation

The RT4/5637gDNA treated with BS was ultrasonically fragmented to simulate cfDNA, and the normal urine cfDNA did not need to be ultrasonically fragmented for single-strand quantification. Gradients are incorporated according to the DNA concentration, and each group of the present application has set up 8 model standard substance gradients (the bladder urothelial carcinoma gDNA content is 100%, 20%, 10%, 5%, 2%, 1%, 0.5%) , 0%).

4. Methylation multiplex PCR, index PCR and 2 recovery (reference embodiment 2)

5. Results

According to the data analysis results (see FIG. 2 ), it can be seen that the present invention can detect tumors well according to key regions. Among them, in the cell line RT4 (non-muscle invasive), this method can correctly distinguish normal urine DNA samples from samples containing tumor DNA when the tumor DNA content is >1%; while in the cell line 5637 (muscle invasive ), when the tumor DNA content > 0.5%, it can be well distinguished from normal urine DNA samples, and the repeatability of 3 times is good.

Example 4

A total of 121 samples were included in this application, including tumor tissue from 27 patients with bladder urothelial carcinoma, preoperative urine from 70 patients with bladder urothelial carcinoma, and urine from 24 normal individuals.

In this application, 121 cases of samples were subjected to methylation multiplex PCR library construction to determine the judgment of the present invention on the distinction between normal people and bladder urothelial carcinoma, non-muscle invasiveness and muscle invasiveness. The specific process is as follows:

1. DNA extraction

The gDNA extraction kit was purchased from Meiji Company (D6315-03) or QIAGEN Company (69506), and was performed according to the kit instructions.

The cfDNA extraction kit was purchased from Zymo (D3061) or Thermo Fisher (A29319), and was performed according to the kit instructions.

2. DNA bisulfite conversion

200ng of tissue gDNA was taken for BS treatment, and 100ng of cfDNA was taken for BS treatment (less than 100ng was supplemented with λDNA). λDNA was purchased from Takala (3010). Specific steps refer to embodiment 2.

3. Methylation multiplex PCR, index PCR and 2 recovery (reference embodiment 2)

4. Construct a generalized linear model for the degree of methylation at each site according to the multiple PCR off-machine data to distinguish between bladder urothelial carcinoma tissue and urine. From this model, other sample results are calculated.

5. Results

According to the analysis results of off-machine data (see Figure 3), this method can well distinguish bladder urothelial carcinoma tissue from normal urine (AUC=1.0). In bladder urothelial carcinoma tissue, muscle invasion and The discrimination of non-muscle invasiveness was also good (AUC=0.969). In addition, in all urine samples, we can also find that the urine of patients with muscle invasion and non-muscle invasion (AUC=0.967), as well as the urine of tumor patients and normal people (AUC=0.94) The discrimination is also better.

The detection sample targeted in this application is urine cfDNA, and we calculated the sensitivity and specificity of the urine sample based on the data, and the specific results are shown in Figure 3. In this application, when using urine to diagnose bladder urothelial carcinoma and normal people, the sensitivity is 0.889, and the specificity is 0.945; For roadside carcinoma, the sensitivity is 0.850 and the specificity is 1.000.

Claims (7)

1. A primer combination for detecting DNA methylation differential regions in samples to be tested is prepared for detecting or predicting whether a subject has bladder urothelial carcinoma or whether the patient with bladder urothelial carcinoma is muscle invasive or non-muscle-invasive reagents or kits, wherein the differential DNA methylation region is selected from one or more regions in the human hg19 version genome as shown below: chr1: 86038429bp-86548655bp, chr7 : 23210805bp-23541085bp, chr12: 50786446bp-51796719bp, chr12: 53396863bp-55397136bp, chr12: 51400340bp-55400617bp, chr5: 2327659bp-239 7867bp, chr5: 5227879bp-5298195bp, chr5: 14341021bp-14541266bp, chr5: 15200260bp-15900461bp, chr8: 2006233bp -2146598bp, chr8: 102136659bp-102436833bp, chr12: 130914602bp-130994836bp, chr17: 77333510bp-77393712bp, chr18: 3869388bp-3899760bp, chr 18: 11741967bp-11762330bp, chr19: 1103012bp-1123479bp and chr3: 180428100bp-1814585400bp.
2. The use according to claim 1, wherein the primer combination comprises BLCBSP_V1-4, BLCBSP_V1-9, BLCBSP_V1-13, BLCBSP_V1-14, BLCBSP_V1-15, BLCBSP_V2-7, BLCBSP_V2-8, BLCBSP_V2-10, BLCBSP_V2-11, BLCBSP_V2-16, BLCBSP_V2-17, BLCBSP_V2-18, BLCBSP_V2-19, BLCBSP_V2-21, BLCBSP_V2-22, BLCBSP_V2-24, BLCBSP_V2-33, BLCBSP_V2-35, MN-DMR4-1; LCBSP_V1- The forward primer sequence among 4 is as shown in SEQ ID NO:1, the reverse primer sequence among BLCBSP_V1-4 is as shown in SEQ ID NO:2; The forward primer sequence among BLCBSP_V1-9 is as shown in SEQ ID NO:3 Shown, the reverse primer sequence in BLCBSP_V1-9 is shown in SEQ ID NO:4; The forward primer sequence in BLCBSP_V1-13 is shown in SEQ ID NO:5, and the reverse primer sequence in BLCBSP_V1-13 is shown in SEQ ID Shown in NO:6; The forward primer sequence among BLCBSP_V1-14 is shown in SEQ ID NO:7, and the reverse primer sequence among BLCBSP_V1-14 is shown in SEQ ID NO:8; The forward primer among BLCBSP_V1-15 The sequence is shown in SEQ ID NO: 9, the reverse primer sequence in BLCBSP_V1-15 is shown in SEQ ID NO: 10; the forward primer sequence in BLCBSP_V2-7 is shown in SEQ ID NO: 11, in BLCBSP_V2-7 The sequence of the reverse primer is shown in SEQ ID NO:12; the sequence of the forward primer in BLCBSP_V2-8 is shown in SEQ ID NO:13, and the sequence of the reverse primer in BLCBSP_V2-8 is shown in SEQ ID NO:14; The forward primer sequence in BLCBSP_V2-10 is shown in SEQ ID NO:15, and the reverse primer sequence in BLCBSP_V2-10 is shown in SEQ ID NO:16; The forward primer sequence in BLCBSP_V2-11 is as shown in SEQ ID NO: 17, the reverse primer sequence in BLCBSP_V2-11 is shown in SEQ ID NO:18; the forward primer sequence in BLCBSP_V2-16 is shown in SEQ ID NO:19, and the reverse primer sequence in BLCBSP_V2-16 is shown in Shown in SEQ ID NO:20; The forward primer sequence in BLCBSP_V2-17 is shown in SEQ ID NO:21, and the reverse primer sequence in BLCBSP_V2-17 is shown in SEQ ID NO:22; The forward primer sequence in BLCBSP_V2-18 To the primer sequence as shown in SEQ ID NO: 23, the reverse primer sequence in BLCBSP_V2-18 is shown in SEQ ID NO: 24; The forward primer sequence in BLCBSP_V2-19 is shown in SEQ ID NO: 25, BLCBSP_V2- The reverse primer sequence in 19 is shown in SEQ ID NO:26; The forward primer sequence in BLCBSP_V2-21 is shown in SEQ ID NO:27, and the reverse primer sequence in BLCBSP_V2-21 is shown in SEQ ID NO:28 Shown; The forward primer sequence in BLCBSP_V2-22 is shown in SEQ ID NO:29, and the reverse primer sequence in BLCBSP_V2-22 is shown in SEQ ID NO:30; The forward primer sequence in BLCBSP_V2-24 is shown in SEQ ID Shown in NO:31, the reverse primer sequence in BLCBSP_V2-24 is shown in SEQ ID NO:32; The forward primer sequence in BLCBSP_V2-33 is shown in SEQ ID NO:33, the reverse primer sequence in BLCBSP_V2-33 The sequence is as shown in SEQ ID NO:34; the forward primer sequence in BLCBSP_V2-35 is as shown in SEQ ID NO:35, and the reverse primer sequence in BLCBSP_V2-35 is as shown in SEQ ID NO:36; MN-DMR4- The forward primer sequence in 1 is shown in SEQ ID NO:37, and the reverse primer sequence in MN-DMR4-1 is shown in SEQ ID NO:38.
3. The use according to claim 1 or 2, wherein the sample to be tested is urine cfDNA or bladder urothelial carcinoma tissue gDNA derived from the subject.
4. A test kit for detecting or predicting whether a subject suffers from bladder urothelial carcinoma or whether the type of bladder urothelial carcinoma is muscle invasive or non-muscle invasive, which includes a test kit for detecting A multiplex PCR reagent for the DNA methylation difference region in the sample, wherein the DNA methylation difference region is selected from one or more regions in the human hg19 version genome as shown below: chr1: 86038429bp-86548655bp, chr7: 23210805bp-23541085bp, chr12: 50786446bp-51796719bp, chr12: 53396863bp-55397136bp, chr12: 51400340bp-55400617bp, chr5: 2327659bp-2397 867bp, chr5: 5227879bp-5298195bp, chr5: 14341021bp-14541266bp, chr5: 15200260bp-15900461bp, chr8: 2006233bp- 2146598bp, chr8: 102136659bp-102436833bp, chr12: 130914602bp-130994836bp, chr17: 77333510bp-77393712bp, chr18: 3869388bp-3899760bp, chr 18: 11741967bp-11762330bp, chr19: 1103012bp-1123479bp and chr3: 180428100bp-1814585400bp.
5. The kit according to claim 4, wherein the multiplex PCR reagents include a primer combination, a positive reference product and a negative reference product.
6. The kit according to claim 5, wherein the primer combination comprises BLCBSP_V1-4, BLCBSP_V1-9, BLCBSP_V1-13, BLCBSP_V1-14, BLCBSP_V1-15, BLCBSP_V2-7, BLCBSP_V2-8, BLCBSP_V2-10 , BLCBSP_V2-11, BLCBSP_V2-16, BLCBSP_V2-17, BLCBSP_V2-18, BLCBSP_V2-19, BLCBSP_V2-21, BLCBSP_V2-22, BLCBSP_V2-24, BLCBSP_V2-33, BLCBSP_V2-35, MN-DMR4-1; , BLCBSP_V1 The forward primer sequence in -4 is shown in SEQ ID NO:1, the reverse primer sequence in BLCBSP_V1-4 is shown in SEQ ID NO:2; The forward primer sequence in BLCBSP_V1-9 is shown in SEQ ID NO:3 Shown, the reverse primer sequence in BLCBSP_V1-9 is shown in SEQ ID NO:4; The forward primer sequence in BLCBSP_V1-13 is shown in SEQ ID NO:5, and the reverse primer sequence in BLCBSP_V1-13 is shown in SEQ ID NO: Shown in ID NO:6; The forward primer sequence in BLCBSP_V1-14 is shown in SEQ ID NO:7, and the reverse primer sequence in BLCBSP_V1-14 is shown in SEQ ID NO:8; The forward primer sequence in BLCBSP_V1-15 The primer sequence is shown in SEQ ID NO:9, the reverse primer sequence in BLCBSP_V1-15 is shown in SEQ ID NO:10; the forward primer sequence in BLCBSP_V2-7 is shown in SEQ ID NO:11, BLCBSP_V2-7 The reverse primer sequence in BLCBSP_V2-8 is shown in SEQ ID NO:12; The forward primer sequence in BLCBSP_V2-8 is shown in SEQ ID NO:13, and the reverse primer sequence in BLCBSP_V2-8 is shown in SEQ ID NO:14 ; The forward primer sequence in BLCBSP_V2-10 is shown in SEQ ID NO:15, and the reverse primer sequence in BLCBSP_V2-10 is shown in SEQ ID NO:16; The forward primer sequence in BLCBSP_V2-11 is shown in SEQ ID NO Shown in: 17, the reverse primer sequence in BLCBSP_V2-11 is shown in SEQ ID NO: 18; The forward primer sequence in BLCBSP_V2-16 is shown in SEQ ID NO: 19, the reverse primer sequence in BLCBSP_V2-16 As shown in SEQ ID NO:20; The forward primer sequence among BLCBSP_V2-17 is as shown in SEQ ID NO:21, and the reverse primer sequence among BLCBSP_V2-17 is as shown in SEQ ID NO:22; Among the BLCBSP_V2-18 The forward primer sequence is shown in SEQ ID NO:23, the reverse primer sequence in BLCBSP_V2-18 is shown in SEQ ID NO:24; the forward primer sequence in BLCBSP_V2-19 is shown in SEQ ID NO:25, BLCBSP_V2 The reverse primer sequence in -19 is shown in SEQ ID NO:26; The forward primer sequence in BLCBSP_V2-21 is shown in SEQ ID NO:27, and the reverse primer sequence in BLCBSP_V2-21 is shown in SEQ ID NO:28 Shown; The forward primer sequence in BLCBSP_V2-22 is shown in SEQ ID NO:29, and the reverse primer sequence in BLCBSP_V2-22 is shown in SEQ ID NO:30; The forward primer sequence in BLCBSP_V2-24 is shown in SEQ ID NO:29 Shown in ID NO:31, the reverse primer sequence in BLCBSP_V2-24 is shown in SEQ ID NO:32; The forward primer sequence in BLCBSP_V2-33 is shown in SEQ ID NO:33, the reverse in BLCBSP_V2-33 The primer sequence is shown in SEQ ID NO:34; The forward primer sequence in BLCBSP_V2-35 is shown in SEQ ID NO:35, and the reverse primer sequence in BLCBSP_V2-35 is shown in SEQ ID NO:36; MN-DMR4 The forward primer sequence in -1 is shown in SEQ ID NO:37, and the reverse primer sequence in MN-DMR4-1 is shown in SEQ ID NO:38.
7. The kit according to any one of claims 4 to 6, wherein the sample to be tested is urine cfDNA or bladder urothelial carcinoma tissue gDNA derived from the subject.

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