CN114107513B - Detection method and kit for bladder urothelial cancer diagnosis - Google Patents

Detection method and kit for bladder urothelial cancer diagnosis Download PDF

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CN114107513B
CN114107513B CN202210097027.1A CN202210097027A CN114107513B CN 114107513 B CN114107513 B CN 114107513B CN 202210097027 A CN202210097027 A CN 202210097027A CN 114107513 B CN114107513 B CN 114107513B
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CN114107513A (en
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张翼
吴凯
单柳颖
金婉
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Ankang Youle Zhongcheng Medical Equipment Co.,Ltd.
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Beijing Youle Fusheng Technology Co ltd
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The invention relates to the field of biotechnology and medical diagnosis, in particular to a detection method and a kit for diagnosing bladder urothelial cancer. The invention provides application of a primer combination for detecting a DNA methylation difference area in a sample to be detected in preparing a reagent or a kit for detecting or predicting whether a subject suffers from bladder urothelial cancer or whether the type of the patient suffering from bladder urothelial cancer is muscle-layer invasive or non-muscle-layer invasive, and a kit for detecting or predicting whether the subject suffers from bladder urothelial cancer or whether the type of the patient suffering from bladder urothelial cancer is muscle-layer invasive or non-muscle-layer invasive. The detection method and the kit for diagnosing the bladder urothelial cancer, provided by the invention, have excellent sensitivity and specificity, have great positive significance for clinical diagnosis and treatment of the bladder urothelial cancer and later-stage monitoring and prognosis evaluation, and provide a theoretical basis for further early diagnosis.

Description

Detection method and kit for bladder urothelial cancer diagnosis
Technical Field
The invention relates to the field of biotechnology and medical diagnosis, in particular to a detection method and a kit for diagnosing bladder urothelial cancer.
Background
Bladder cancer (Bladder cancer, BCa) is the most common malignancy of the urinary system, with a worldwide increasing incidence. BCa fall into three main pathological categories: bladder Urothelial Carcinoma (BLCA), squamous cell Carcinoma and adenocarcinoma, with BLCA accounting for 90%, with 212,500 new deaths worldwide in 2020.
BLCA can be further divided into muscular layer invasiveness BCa (muscular layer invasiveness) and non-muscular layer invasiveness BCa (non-muscular layer infiltrates), with non-muscular layer infiltrates accounting for approximately 75% of all cases. The biological behaviors of the two types of BLCA are different, and the prognosis is very different, and needs to be treated differently. Due to non-muscle layer infiltration and bladder muscle layer, effective treatment can be achieved by urethral cystectomy in combination with intracapsular perfusion of chemotherapeutic drugs or BCG. The muscle layer infiltrative property is more invasive, the growth speed is high, the metastatic potential is greatly improved, and bladder excision treatment is usually needed. However, approximately 70% of clinically diagnosed non-muscle infiltrates do not respond fully to standard practice, and eventually relapse even translates into muscle infiltrates. Surgical delays resulting from inaccurate initial pathological diagnosis can result in a 50% increase in the overall mortality risk ratio. Therefore, the method has important clinical significance for early diagnosis of bladder urothelial cancer.
Currently, the main clinical diagnosis methods of bladder urothelial cancer include cystoscopy, urine cast-off cytology, B-ultrasound, CT scan, etc. Cystoscopy is considered as the gold standard for diagnosis of bladder urothelial cancer, but this examination method is invasive and may cause complications such as urinary tract infection, urinary tract injury, bladder injury, and the like. Urine apheresis cytology, such as Fluorescence In Situ Hybridization (FISH) detection, can aid in the diagnosis of BCa, but overall sensitivity and specificity are unsatisfactory, 50% and 85%, respectively. Therefore, it is necessary to develop a new noninvasive bladder urothelial cancer detection scheme with higher sensitivity, stronger specificity and shorter detection period. Particularly, the bladder urothelial cancer has the characteristics of unobvious early symptoms, rapid development after disease onset, difficult clear diagnosis of pathology and the like, and the increase of the detection rate and the shortening of the detection time are particularly critical.
Disclosure of Invention
Problems to be solved by the invention
The invention provides a detection method and a kit for diagnosing bladder urothelial cancer, aiming at solving the problems that the detection is difficult because early symptoms of bladder urothelial cancer are not obvious, excellent detection methods such as a bladder endoscope are invasive and easily cause complications, and the existing nondestructive detection method has low sensitivity and specificity.
Means for solving the problems
[1] Use of a primer combination for detecting a DNA methylation difference region in a test sample for the preparation of a reagent or kit for detecting or predicting whether a subject has urothelial bladder cancer or whether a subject is typed for muscle-invasive or non-muscle-invasive, wherein the DNA methylation difference region is selected from one or more of the regions of the human hg19 version of the genome as shown below: chr 1: 86038429bp-86548655bp, chr 7: 23210805bp-23541085bp, chr 12: 50786446bp-51796719bp, chr 12: 53396863bp-55397136bp, chr 12: 51400340bp-55400617bp, chr 5: 2327659bp-2397867bp, chr 5: 5227879bp-5298195bp, chr 5: 14341021bp-14541266bp, chr 5: 15200260bp-15900461bp, chr 8: 2006233bp-2146598bp, chr 8: 102136659bp-102436833bp, chr 12: 130914602bp-130994836bp, chr 17: 77333510bp-77393712bp, chr 18: 3869388bp-3899760bp, chr 18: 11741967bp-11762330bp, chr 19: 1103012bp-1123479bp and chr 3: 180428100bp-181458540 bp.
[2] The use according to [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-DMR 4-1; wherein, the sequence of the forward primer in BLCBSP _ V1-4 is shown as SEQ ID NO. 1, and the sequence of the reverse primer in BLCBSP _ V1-4 is shown as SEQ ID NO. 2; the sequence of the forward primer in BLCBSP _ V1-9 is shown as SEQ ID NO. 3, and the sequence of the reverse primer in BLCBSP _ V1-9 is shown as SEQ ID NO. 4; the forward primer sequence in BLCBSP _ V1-13 is shown as SEQ ID NO. 5, and the reverse primer sequence in BLCBSP _ V1-13 is shown as SEQ ID NO. 6; the forward primer sequence in BLCBSP _ V1-14 is shown as SEQ ID NO. 7, and the reverse primer sequence in BLCBSP _ V1-14 is shown as SEQ ID NO. 8; the forward primer sequence in BLCBSP _ V1-15 is shown as SEQ ID NO. 9, and the reverse primer sequence in BLCBSP _ V1-15 is shown as SEQ ID NO. 10; the sequence of the forward primer in BLCBSP _ V2-7 is shown as SEQ ID NO. 11, and the sequence of the reverse primer in BLCBSP _ V2-7 is shown as SEQ ID NO. 12; the sequence of the forward primer in BLCBSP _ V2-8 is shown as SEQ ID NO. 13, and the sequence of the reverse primer in BLCBSP _ V2-8 is shown as SEQ ID NO. 14; the forward primer sequence in BLCBSP _ V2-10 is shown as SEQ ID NO. 15, and the reverse primer sequence in BLCBSP _ V2-10 is shown as SEQ ID NO. 16; the sequence of the forward primer in BLCBSP _ V2-11 is shown as SEQ ID NO. 17, and the sequence of the reverse primer in BLCBSP _ V2-11 is shown as SEQ ID NO. 18; the sequence of a forward primer in BLCBSP _ V2-16 is shown as SEQ ID NO. 19, and the sequence of a reverse primer in BLCBSP _ V2-16 is shown as SEQ ID NO. 20; the forward primer sequence in BLCBSP _ V2-17 is shown as SEQ ID NO:21, and the reverse primer sequence in BLCBSP _ V2-17 is shown as SEQ ID NO: 22; the forward primer sequence in BLCBSP _ V2-18 is shown as SEQ ID NO. 23, and the reverse primer sequence in BLCBSP _ V2-18 is shown as SEQ ID NO. 24; the forward primer sequence in BLCBSP _ V2-19 is shown as SEQ ID NO. 25, and the reverse primer sequence in BLCBSP _ V2-19 is shown as SEQ ID NO. 26; the forward primer sequence in BLCBSP _ V2-21 is shown as SEQ ID NO. 27, and the reverse primer sequence in BLCBSP _ V2-21 is shown as SEQ ID NO. 28; the forward primer sequence in BLCBSP _ V2-22 is shown as SEQ ID NO. 29, and the reverse primer sequence in BLCBSP _ V2-22 is shown as SEQ ID NO. 30; the forward primer sequence in BLCBSP _ V2-24 is shown as SEQ ID NO. 31, and the reverse primer sequence in BLCBSP _ V2-24 is shown as SEQ ID NO. 32; the forward primer sequence in BLCBSP _ V2-33 is shown as SEQ ID NO. 33, and the reverse primer sequence in BLCBSP _ V2-33 is shown as SEQ ID NO. 34; the forward primer sequence in BLCBSP _ V2-35 is shown as SEQ ID NO. 35, and the reverse primer sequence in BLCBSP _ V2-35 is shown as SEQ ID NO. 36; the forward primer sequence in MN-DMR4-1 is shown as SEQ ID NO:37, and the reverse primer sequence in MN-DMR4-1 is shown as SEQ ID NO: 38.
[3] The use according to [1] or [2], wherein the sample to be tested is urine cfDNA or bladder urothelial cancer tissue gDNA derived from the subject.
[4] A kit for detecting or predicting whether a subject has urothelial carcinoma of the bladder or whether a patient with urothelial carcinoma of the bladder is typed for muscle-or non-muscle-invasive comprising multiplex PCR reagents for detecting regions of DNA methylation differentiation in a test sample, wherein the regions of DNA methylation differentiation are selected from one or more of the regions of the human hg19 version of the genome as shown below: chr 1: 86038429bp-86548655bp, chr 7: 23210805bp-23541085bp, chr 12: 50786446bp-51796719bp, chr 12: 53396863bp-55397136bp, chr 12: 51400340bp-55400617bp, chr 5: 2327659bp-2397867bp, chr 5: 5227879bp-5298195bp, chr 5: 14341021bp-14541266bp, chr 5: 15200260bp-15900461bp, chr 8: 2006233bp-2146598bp, chr 8: 102136659bp-102436833bp, chr 12: 130914602bp-130994836bp, chr 17: 77333510bp-77393712bp, chr 18: 3869388bp-3899760bp, chr 18: 11741967bp-11762330bp, chr 19: 1103012bp-1123479bp and chr 3: 180428100bp-181458540 bp.
[5] The kit according to [4], wherein the multiplex PCR reagent comprises a primer combination, a positive reference substance and a negative reference substance.
[6] The kit of [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, BLSP _ V2-24, BLCBSP _ V2-33, BLCBSP _ V2-35, MN-DMR 4-1; wherein, the sequence of the forward primer in BLCBSP _ V1-4 is shown as SEQ ID NO. 1, and the sequence of the reverse primer in BLCBSP _ V1-4 is shown as SEQ ID NO. 2; the sequence of the forward primer in BLCBSP _ V1-9 is shown as SEQ ID NO. 3, and the sequence of the reverse primer in BLCBSP _ V1-9 is shown as SEQ ID NO. 4; the forward primer sequence in BLCBSP _ V1-13 is shown as SEQ ID NO. 5, and the reverse primer sequence in BLCBSP _ V1-13 is shown as SEQ ID NO. 6; the forward primer sequence in BLCBSP _ V1-14 is shown as SEQ ID NO. 7, and the reverse primer sequence in BLCBSP _ V1-14 is shown as SEQ ID NO. 8; the forward primer sequence in BLCBSP _ V1-15 is shown as SEQ ID NO. 9, and the reverse primer sequence in BLCBSP _ V1-15 is shown as SEQ ID NO. 10; the sequence of the forward primer in BLCBSP _ V2-7 is shown as SEQ ID NO. 11, and the sequence of the reverse primer in BLCBSP _ V2-7 is shown as SEQ ID NO. 12; the sequence of a forward primer in BLCBSP _ V2-8 is shown as SEQ ID NO. 13, and the sequence of a reverse primer in BLCBSP _ V2-8 is shown as SEQ ID NO. 14; the forward primer sequence in BLCBSP _ V2-10 is shown as SEQ ID NO. 15, and the reverse primer sequence in BLCBSP _ V2-10 is shown as SEQ ID NO. 16; the sequence of the forward primer in BLCBSP _ V2-11 is shown as SEQ ID NO. 17, and the sequence of the reverse primer in BLCBSP _ V2-11 is shown as SEQ ID NO. 18; the forward primer sequence in BLCBSP _ V2-16 is shown as SEQ ID NO. 19, and the reverse primer sequence in BLCBSP _ V2-16 is shown as SEQ ID NO. 20; the forward primer sequence in BLCBSP _ V2-17 is shown as SEQ ID NO:21, and the reverse primer sequence in BLCBSP _ V2-17 is shown as SEQ ID NO: 22; the forward primer sequence in BLCBSP _ V2-18 is shown as SEQ ID NO. 23, and the reverse primer sequence in BLCBSP _ V2-18 is shown as SEQ ID NO. 24; the forward primer sequence in BLCBSP _ V2-19 is shown as SEQ ID NO. 25, and the reverse primer sequence in BLCBSP _ V2-19 is shown as SEQ ID NO. 26; the sequence of a forward primer in BLCBSP _ V2-21 is shown as SEQ ID NO. 27, and the sequence of a reverse primer in BLCBSP _ V2-21 is shown as SEQ ID NO. 28; the forward primer sequence in BLCBSP _ V2-22 is shown as SEQ ID NO. 29, and the reverse primer sequence in BLCBSP _ V2-22 is shown as SEQ ID NO. 30; the forward primer sequence in BLCBSP _ V2-24 is shown as SEQ ID NO. 31, and the reverse primer sequence in BLCBSP _ V2-24 is shown as SEQ ID NO. 32; the forward primer sequence in BLCBSP _ V2-33 is shown as SEQ ID NO. 33, and the reverse primer sequence in BLCBSP _ V2-33 is shown as SEQ ID NO. 34; the forward primer sequence in BLCBSP _ V2-35 is shown as SEQ ID NO. 35, and the reverse primer sequence in BLCBSP _ V2-35 is shown as SEQ ID NO. 36; the forward primer sequence in MN-DMR4-1 is shown as SEQ ID NO:37, and the reverse primer sequence in MN-DMR4-1 is shown as 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 cancer tissue gDNA derived from the subject.
[8] A method of detecting or predicting whether a subject has urothelial carcinoma of the bladder or whether a patient with urothelial carcinoma of the bladder is typed for muscle-invasive or non-muscle-invasive, comprising the steps of: extracting the urine cfDNA of a subject, performing bisulfite conversion on the urine cfDNA, performing multiplex PCR by using a primer combination, recovering products, performing index PCR and high-throughput sequencing on the products of the multiplex PCR, calculating the methylation level of a DNA methylation difference region between bladder muscle invasive urothelial cancer, bladder non-muscle invasive urothelial cancer and normal bladder tissues, and judging whether the subject suffers from bladder urinary epithelial cancer or whether the type of the patient suffering from bladder urinary epithelial cancer is muscle invasive or non-muscle invasive; wherein the content of the first and second substances,
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, and MN-DMR 4-1; wherein, the sequence of the forward primer in BLCBSP _ V1-4 is shown as SEQ ID NO. 1, and the sequence of the reverse primer in BLCBSP _ V1-4 is shown as SEQ ID NO. 2; the sequence of the forward primer in BLCBSP _ V1-9 is shown as SEQ ID NO. 3, and the sequence of the reverse primer in BLCBSP _ V1-9 is shown as SEQ ID NO. 4; the sequence of a forward primer in BLCBSP _ V1-13 is shown as SEQ ID NO. 5, and the sequence of a reverse primer in BLCBSP _ V1-13 is shown as SEQ ID NO. 6; the forward primer sequence in BLCBSP _ V1-14 is shown as SEQ ID NO. 7, and the reverse primer sequence in BLCBSP _ V1-14 is shown as SEQ ID NO. 8; the forward primer sequence in BLCBSP _ V1-15 is shown as SEQ ID NO. 9, and the reverse primer sequence in BLCBSP _ V1-15 is shown as SEQ ID NO. 10; the sequence of the forward primer in BLCBSP _ V2-7 is shown as SEQ ID NO. 11, and the sequence of the reverse primer in BLCBSP _ V2-7 is shown as SEQ ID NO. 12; the sequence of the forward primer in BLCBSP _ V2-8 is shown as SEQ ID NO. 13, and the sequence of the reverse primer in BLCBSP _ V2-8 is shown as SEQ ID NO. 14; the forward primer sequence in BLCBSP _ V2-10 is shown as SEQ ID NO. 15, and the reverse primer sequence in BLCBSP _ V2-10 is shown as SEQ ID NO. 16; the sequence of the forward primer in BLCBSP _ V2-11 is shown as SEQ ID NO. 17, and the sequence of the reverse primer in BLCBSP _ V2-11 is shown as SEQ ID NO. 18; the forward primer sequence in BLCBSP _ V2-16 is shown as SEQ ID NO. 19, and the reverse primer sequence in BLCBSP _ V2-16 is shown as SEQ ID NO. 20; the forward primer sequence in BLCBSP _ V2-17 is shown as SEQ ID NO:21, and the reverse primer sequence in BLCBSP _ V2-17 is shown as SEQ ID NO: 22; the forward primer sequence in BLCBSP _ V2-18 is shown as SEQ ID NO. 23, and the reverse primer sequence in BLCBSP _ V2-18 is shown as SEQ ID NO. 24; the forward primer sequence in BLCBSP _ V2-19 is shown as SEQ ID NO. 25, and the reverse primer sequence in BLCBSP _ V2-19 is shown as SEQ ID NO. 26; the forward primer sequence in BLCBSP _ V2-21 is shown as SEQ ID NO. 27, and the reverse primer sequence in BLCBSP _ V2-21 is shown as SEQ ID NO. 28; the forward primer sequence in BLCBSP _ V2-22 is shown as SEQ ID NO. 29, and the reverse primer sequence in BLCBSP _ V2-22 is shown as SEQ ID NO. 30; the forward primer sequence in BLCBSP _ V2-24 is shown as SEQ ID NO. 31, and the reverse primer sequence in BLCBSP _ V2-24 is shown as SEQ ID NO. 32; the forward primer sequence in BLCBSP _ V2-33 is shown as SEQ ID NO. 33, and the reverse primer sequence in BLCBSP _ V2-33 is shown as SEQ ID NO. 34; the forward primer sequence in BLCBSP _ V2-35 is shown as SEQ ID NO. 35, and the reverse primer sequence in BLCBSP _ V2-35 is shown as SEQ ID NO. 36; the forward primer sequence in the MN-DMR4-1 is shown as SEQ ID NO. 37, and the reverse primer sequence in the MN-DMR4-1 is shown as SEQ ID NO. 38;
the DNA methylation difference region is selected from one or more regions in the human hg19 version genome as shown below: chr 1: 86038429bp-86548655bp, chr 7: 23210805bp-23541085bp, chr 12: 50786446bp-51796719bp, chr 12: 53396863bp-55397136bp, chr 12: 51400340bp-55400617bp, chr 5: 2327659bp-2397867bp, chr 5: 5227879bp-5298195bp, chr 5: 14341021bp-14541266bp, chr 5: 15200260bp-15900461bp, chr 8: 2006233bp-2146598bp, chr 8: 102136659bp-102436833bp, chr 12: 130914602bp-130994836bp, chr 17: 77333510bp-77393712bp, chr 18: 3869388bp-3899760bp, chr 18: 11741967bp-11762330bp, chr 19: 1103012bp-1123479bp and chr 3: 180428100bp-181458540 bp.
ADVANTAGEOUS EFFECTS OF INVENTION
Through the implementation of the technical scheme, the detection method and the kit for diagnosing the urinary bladder urothelial cancer, provided by the invention, have excellent sensitivity and specificity. The experimental data show that when the urine is used for diagnosing whether the subject has bladder urothelial carcinoma, the sensitivity is 0.889, and the specificity is 0.945; when urine was used to distinguish and classify the muscular-invasive bladder urothelial cancer and non-muscular-invasive bladder urothelial cancer, the sensitivity was 0.850 and the specificity was 1.000. Has great positive significance for the diagnosis and treatment of bladder urothelial cancer, the monitoring and the prognosis evaluation in the later period and provides a theoretical basis for further early diagnosis.
Drawings
FIG. 1 is a plot of methylation levels of model standards.
FIG. 2 shows the result of detecting tumor DNA of bladder urothelial carcinoma cell lines RT4 and 5637.
FIG. 3 shows the results of DNA detection of tumor in tissues and urine of 121 real samples.
Detailed Description
The following describes embodiments of the present invention, but the present invention is not limited to these embodiments. The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the claims, and embodiments and examples obtained by appropriately combining the technical means disclosed in the respective embodiments and examples are also included in the technical scope of the present invention.
Unless defined otherwise, 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 terms "a" or "an" or "the" may mean "one" or "one" and may also mean "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.
In the present invention, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.
Although the disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in this disclosure means "and/or" unless expressly specified to the contrary only as an alternative or alternative.
In the present invention, unless otherwise specified, the position numbering of human chromosomes in the present invention is based on hg19 version.
In the invention, the principle of DNA bisulfite conversion is that a sample to be detected is treated by bisulfite, C base which is not methylated in a genome is converted into U, the U is converted into T after PCR amplification, the T is distinguished from C base which originally has methylation modification, and then whether CpG/CHG/CHH sites are methylated or not can be judged by combining a high-throughput sequencing technology and comparing the T with a reference sequence.
In the present invention, "cfDNA" is Cell-Free Circulating DNA (Cell-Free Circulating DNA) and is partially degraded extracellularly and is an endogenous DNA present in human Circulating blood, urine and other body fluids. The length of cfDNA is usually 200bp or less.
In the present invention, "gDNA" is "genomic DNA", and means the entire DNA content of an organism in a haploid state.
In the present invention, "DNA methylation" refers to a process of transferring a methyl group to a specific base in an organism by using S-adenosylmethionine (SAM) as a methyl donor under the catalysis of DNA methyltransferase. In mammals, methylation of the 5' C-terminus of the nucleotide cytosine residue is predominant. On the human genome, a large amount of DNA methylation occurs at cytosine in CpG dinucleotides, C is cytosine, G is guanine, and p is a phosphate group. DNA methylation can also occur in cytosine in CHG and CHH nucleotide sequences, where H is adenine, cytosine or thymine. DNA methylation may also occur on non-cytosines, such as N6-methyladenine. In addition, DNA methylation may be in the form of 5-hydroxymethylcytosine or the like.
In the present invention, "DNA methylation difference region" and "DNA methylation modification difference region" are used interchangeably and refer to a genomic region composed of a plurality of adjacent base positions in which the degree of DNA methylation modification differs between two biological samples.
In the present invention, "DNA methylation modification" may be expressed as single-base methylation modification, as an average of a plurality of base methylation modifications over a continuous region, as methylation haplotype, or methylation haplotype.
In the present invention, "degree of DNA methylation modification", "level of DNA methylation modification" and "methylation level" are used interchangeably and can be described in terms of the global quantitative parameter β or abundance of methylated haplotypes.
In the present invention, the quantitative parameter β of the DNA methylation modification level as a whole can be expressed as the average methylation modification degree of C base, namely: β ═ the number of C carrying a methylation modification/(the number of C carrying a methylation modification + the number of C not carrying a methylation modification).
In the present invention, the "methylation haplotype" is also referred to as "methylation haplotype" and refers to a combination of methylation modifications at two or more consecutive C base sites on a single DNA fragment. Because the methylation modification of DNA has relevance on the same chromosome (DNA chain), a plurality of adjacent C base sites can exist, and the methylation modification situations are mutually related to form a certain fixed form. Thus, a DNA methylation haplotype is a linkage pattern in which DNA methylation modifications occur on the same chromosome.
In the present invention, the term "abundance of methylated haplotypes" refers to the ratio of a specific methylated haplotype obtained by statistically analyzing methylated haplotype information. Namely: the abundance of a methylation haplotype is the number of DNA fragments that fit into a particular methylation haplotype/total number of DNA fragments that completely cover the genomic region of the methylation haplotype.
In some embodiments, the invention provides a region of bladder urothelial cancer patient with significant differences in DNA methylation from normal human, and a region of DNA differential methylation between the muscle and non-muscle infiltrates of bladder urothelial cancer, in particular, the region of DNA differential methylation is selected from one or more of the regions of the human hg19 version of the genome shown below: chr 1: 86038429bp-86548655bp, chr 7: 23210805bp-23541085bp, chr 12: 50786446bp-51796719bp, chr 12: 53396863bp-55397136bp, chr 12: 51400340bp-55400617bp, chr 5: 2327659bp-2397867bp, chr 5: 5227879bp-5298195bp, chr 5: 14341021bp-14541266bp, chr 5: 15200260bp-15900461bp, chr 8: 2006233bp-2146598bp, chr 8: 102136659bp-102436833bp, chr 12: 130914602bp-130994836bp, chr 17: 77333510bp-77393712bp, chr 18: 3869388bp-3899760bp, chr 18: 11741967bp-11762330bp, chr 19: 1103012bp-1123479bp and chr 3: 180428100bp-181458540 bp.
In the present invention, "multiplex PCR" is also called multiplex primer PCR or multiplex PCR, which is a PCR reaction in which two or more pairs of primers are added to the same PCR reaction system to simultaneously amplify a plurality of nucleic acid fragments, and the reaction principle, reaction reagents and operation process are the same as those of general PCR.
In some specific embodiments, the invention provides a multiplex PCR primer combination designed for the region with obvious DNA methylation difference from normal human patients with bladder urothelial cancer and the region with DNA methylation difference between the muscle layer infiltrability and the non-muscle layer infiltrability of the bladder urothelial cancer.
In some preferred embodiments, the multiplex PCR primer combinations provided herein include 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, BLSP _ V2-24, BLCBSP _ V2-33, BLCBSP _ V2-35, MN-DMR 4-1; wherein, the forward primer sequence in BLCBSP _ V1-4 is 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGTTGGATGTTTGAGTGTGAA-3' (SEQ ID NO:1), and the reverse primer sequence in BLCBSP _ V1-4 is 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCTTAAAAAATATCTCCCCATCT-3' (SEQ ID NO: 2); the forward primer sequence in BLCBSP _ V1-9 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGGAGTGGTGGGGGATG-3' (SEQ ID NO:3), and the reverse primer sequence in BLCBSP _ V1-9 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCTCATTTATCCTAAAACCTTTTC-3' (SEQ ID NO: 4); the forward primer sequence in BLCBSP _ V1-13 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGGTAGGGTTGGAATGAGAA-3' (SEQ ID NO:5), and the reverse primer sequence in BLCBSP _ V1-13 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCTCTATTACTAAAACCCCAAAA-3' (SEQ ID NO: 6); the forward primer sequence in BLCBSP _ V1-14 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGTGAGGGTTGGGGATTATG-3' (SEQ ID NO:7), and the reverse primer sequence in BLCBSP _ V1-14 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCAAAACTATTCTACCATAACAAAC-3' (SEQ ID NO: 8); the forward primer sequence in BLCBSP _ V1-15 is 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNTGAGGGATTTTTAAAAGGGG-3' (SEQ ID NO:9), and the reverse primer sequence in BLCBSP _ V1-15 is 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNACCCTACCAAAAAACTATTCTT-3' (SEQ ID NO: 10); the forward primer sequence in BLCBSP _ V2-7 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGGTTTAGTTTTTAGATATTAGAAG-3' (SEQ ID NO:11), and the reverse primer sequence in BLCBSP _ V2-7 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNAAACTAACCCTATAACCCCA-3' (SEQ ID NO: 12); the forward primer sequence in BLCBSP _ V2-8 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNTTTTATATAAAATTTTTGTGGATGG-3' (SEQ ID NO:13), and the reverse primer sequence in BLCBSP _ V2-8 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNACCTAAATACTTTAAAACTAACCT-3' (SEQ ID NO: 14); the forward primer sequence in BLCBSP _ V2-10 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGTTTGGGTATTTTTATTTGTGAAA-3' (SEQ ID NO:15), and the reverse primer sequence in BLCBSP _ V2-10 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCAAAAACAAACACACAAACTAAC-3' (SEQ ID NO: 16); the forward primer sequence in BLCBSP _ V2-11 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGGGGATTYGTAATAAGTGG-3' (SEQ ID NO:17), and the reverse primer sequence in BLCBSP _ V2-11 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCRAACAAACCCCTAAACTC-3' (SEQ ID NO: 18); the forward primer sequence in BLCBSP _ V2-16 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGTGTGGTTTGAGTGTTTGTT-3' (SEQ ID NO:19), and the reverse primer sequence in BLCBSP _ V2-16 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNAATCCTTCACTCAATCCCAC-3' (SEQ ID NO: 20); the forward primer sequence in BLCBSP _ V2-17 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGGAAATTATTTTGGGGATTGAG-3' (SEQ ID NO:21), and the reverse primer sequence in BLCBSP _ V2-17 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNTAAACCAAATATTTCAAAAAAAACC-3' (SEQ ID NO: 22); the forward primer sequence in BLCBSP _ V2-18 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNAGGAAGGGGTTTTAGTTAAAG-3' (SEQ ID NO:23), and the reverse primer sequence in BLCBSP _ V2-18 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCCTAAAACAAAAATCAAAATATCC-3' (SEQ ID NO: 24); the forward primer sequence in BLCBSP _ V2-19 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGGTAGGAGAAGGTTATTTATG-3' (SEQ ID NO:25), and the reverse primer sequence in BLCBSP _ V2-19 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCTTTTTAAAATATAACACCCATCC-3' (SEQ ID NO: 26); the forward primer sequence in BLCBSP _ V2-21 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGAGGAATTTTTTTTTGGTAGGA-3' (SEQ ID NO:27), and the reverse primer sequence in BLCBSP _ V2-21 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCCCTAATTACAATACTAACACT-3' (SEQ ID NO: 28); the forward primer sequence in BLCBSP _ V2-22 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGATTTGGTGAAGAGTTTTTGG-3' (SEQ ID NO:29), and the reverse primer sequence in BLCBSP _ V2-22 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNTAACTATCACACCCTACAATAC-3' (SEQ ID NO: 30); the forward primer sequence in BLCBSP _ V2-24 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGTTGAGYGAGGTTTGGAG-3' (SEQ ID NO:31), and the reverse primer sequence in BLCBSP _ V2-24 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNAAATCTACTAAATAAATACAACACA-3' (SEQ ID NO: 32); the forward primer sequence in BLCBSP _ V2-33 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNTGTTGGGAAAGGGAGGG-3' (SEQ ID NO:33), and the reverse primer sequence in BLCBSP _ V2-33 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNCCCAACTTCACTTTTTCACT-3' (SEQ ID NO: 34); the forward primer sequence in BLCBSP _ V2-35 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNTGGGGTTTAGAGGGATGG-3' (SEQ ID NO:35), and the reverse primer sequence in BLCBSP _ V2-35 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNTCTAATAACCCCTACTAATCAC-3' (SEQ ID NO: 36); the forward primer sequence in MN-DMR4-1 was 5 '-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNNGTTAGGGTTTGTAGGGTTT-3' (SEQ ID NO:37) and the reverse primer sequence in MN-DMR4-1 was 5 '-TTAATGCAACGATCGTCGAAATTCGCNNNNNNNTAATAAAACTAAACTACACTAACCTC-3' (SEQ ID NO: 38).
To more clearly illustrate the technical solutions of the present invention, the following embodiments are further described, but the present invention is not limited thereto, and these embodiments are only some examples of the present invention. Unless otherwise indicated, instruments, reagents, materials, laboratory animals and the like used in the present invention are commercially available in a conventional manner.
Example 1
The invention is based on the single cell transcriptome, the single cell chromatin accessibility sequencing and the whole genome DNA methylation sequencing of bladder muscle layer invasive urothelial carcinoma (MIBC), bladder non-muscle layer invasive urothelial carcinoma (NMIBC) and normal bladder tissues, and analyzes and finds that cells of different origin types have different DNA methylation characteristics, so that the opening degree of chromatin is different, the difference of gene expression is influenced, and finally the cells generate different fates, some are normal cells, and some develop into muscle layer invasive cancer cells or non-muscle layer invasive cancer cells. By statistical analysis of these DNA methylation profiles, regions with significant differences in methylation levels (DMR) in the three source cells were selected as shown in table 1. The invention is based on a methylation multiplex PCR method, the DMR regions are amplified, and the methylation level of the DNA regions is detected through high-throughput sequencing, wherein the specific primer combination is shown in a table 2:
TABLE 1 detection of methylation differential regions of bladder urothelial cancer
DMR ID NO. Chromosome numbering 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 180428100-181458540 hg19
TABLE 2 primer combinations for methylation multiplex PCR
Figure GDA0003556766790000121
All primers were provided with a linker universal sequence and a single molecular tag sequence consisting of 7 bases:
forward primer adaptor universal sequence:
5’-TTTCCCTACACGACGCTCTTCCGATCTNNNNNNN，
reverse primer linker universal sequence:
5’-TTAATGCAACGATCGTCGAAATTCGCNNNNNNN。
the primers used in the present invention were purchased from Biotechnology (Shanghai) GmbH.
Example 2
Detection of 17 methylated regions was performed using commercial complete methylation and non-methylation pattern standards (available from Zymo corporation).
The specific process is as follows:
DNA bisulfite conversion (BS treatment)
DNA bisulfite conversion kits were purchased from Zymo, Inc., according to kit instructions, and the conversion products were NF-H2And eluting for later use.
2. Modal standard gradient incorporation
And (3) carrying out single-chain quantification after completely methylating the BS after treatment and ultrasonically cutting off the non-methylated mode standard substance. Gradient incorporation was performed according to DNA concentration, and a total of 13 model standard gradients (methylation levels of 100%, 99%, 98%, 95%, 90%, 80%, 50%, 20%, 10%, 5%, 2%, 1%, 0%) were set up.
3. Preparation of methylated multiplex PCR primer combination
The 19 pairs of primers were mixed for use.
4, preparing PCR premix solution
The premix was prepared according to table 3. Reagents for multiplex PCR reactions were purchased from Thermo Fisher (N8080241).
TABLE 3 methylation multiplex PCR premix protocol
Reagent Volume (μ L)
DNA (20ng initial amount) X
10×Buffer 2
10mM dNTPs 0.4
25mM MgCl2 1.2
AmplitaqGoldtaq 0.1
Primer and method for producing the same 4.6
NF-H2O 11.7-X
Total amount of 20
PCR reaction procedure: 10min at 95 ℃; 25 × (95 ℃ 30s, 65 ℃ 30s, 54 ℃ 2min, 65 ℃ 30s, 72 ℃ 30 s); 10min at 72 ℃; storing at 16 ℃.
The reaction product can be stored temporarily at-20 ℃.
5. Multiplex PCR product recovery
1) The product in the PCR tube was transiently pipetted into a corresponding 1.5mL centrifuge tube with 24. mu.L of XP Beads added.
2) Vortex, mix well for 15s, then isolate for 3s, incubate at room temperature for 5 min.
3) After the incubation was completed, the 1.5mL centrifuge tube was placed on a magnetic rack, and left to stand until the liquid was clear (about 5min), and the supernatant was discarded.
4) Adding 180 μ L of freshly prepared 80% ethanol, inverting each of the two sides for 2 times, incubating on a magnetic frame at room temperature for 30s, and discarding the supernatant.
5) Repeating the step 4) once.
6) The 1.5mL centrifuge tube was removed from the magnetic rack and centrifuged for 30 s. The centrifuge tube (1.5 mL) was returned to the magnetic stand, allowed to stand for 1min, and the residue was removed by pipetting with a 20. mu.L pipette.
7) The 1.5mL centrifuge tube lid was opened and the beads were air dried at room temperature until the surface of the beads was matte (about 1-3 min).
8) The 1.5mL centrifuge tube was removed from the magnetic stand and 25. mu.L NF-H was added2And O. Flicking with finger, vortexing, mixing, and incubating at room temperature for 5 min.
9) After the incubation is finished, placing a 1.5mL centrifuge tube on a magnetic frame, and standing until the liquid is clear for later use.
6.index PCR
Reagents for index PCR were purchased from KAPA Biosystems (KK 2602).
The reaction system is shown in Table 4:
TABLE 4 index PCR reaction System
Reagent Volume (μ L)
Recovering the product of the previous step 23
2×PCR mix 25
i5 index 1
i7 index 1
Total amount of 50
PCR reaction procedure: 45s at 98 ℃; 13 × (98 ℃ C. for 15s, 60 ℃ C. for 30s, 72 ℃ C. for 30 s); 5min at 72 ℃; storing at 4 ℃.
The reaction product can be placed at-20 ℃ and recovered the next day.
Index PCR product recovery and quantitation
1) The product in the PCR tube was transiently pipetted into a corresponding 1.5mL centrifuge tube with 60. mu.L of XP Beads added.
2) Vortex, mix well for 15s, then isolate for 3s, incubate at room temperature for 5 min.
3) After incubation, the 1.5mL centrifuge tube was placed on a magnetic rack, and allowed to stand until the liquid cleared (about 5min), and the supernatant was discarded.
4) Adding 180. mu.L of freshly prepared 80% ethanol, inverting each of the two sides for 2 times, incubating the mixture on a magnetic frame at room temperature for 30s, and discarding the supernatant.
5) Repeating the step 4) once.
6) The 1.5mL centrifuge tube was removed from the magnetic rack and centrifuged for 30 s. The centrifuge tube (1.5 mL) was returned to the magnetic stand, allowed to stand for 1min, and the residue was removed by pipetting with a 20. mu.L pipette.
7) The 1.5mL centrifuge tube lid was opened and the beads were air dried at room temperature until the surface of the beads was matte (about 1-3 min).
8) A1.5 mL centrifuge tube was removed from the magnetic rack and 36. mu.L of EB was added. Flicking with a finger, mixing by Vortex, and incubating for 5min at room temperature.
9) After incubation, the 1.5mL centrifuge tube was placed on a magnetic rack, allowed to stand until the liquid was clear (about 5min), and transferred to a new 1.5mL centrifuge tube.
10) Double strand quantification was performed using dsDNA Qubit.
8. Library computer
After the concentration of the library is determined by fluorescent quantitative PCR and qualified, PE150 sequencing is carried out by using an Illumina Novaseq platform, and the data volume of each library is 5-8M reads.
9. Results
The methylation level discrimination of the 13 detected pattern standard products with different gradients is good, 1% methylation difference of most sites in hypermethylated or hypomethylated DNA templates can be well identified, and the repeatability of 3 times is good. FIG. 1 is a plot of methylation levels of 13 standards of different gradient patterns after amplification of 19 primer pairs (17 regions).
Example 3
Biological standard substance detection is carried out by using bladder urothelial carcinoma cell strains RT4 and 5637 so as to determine the lowest content detection limit of the cell strains on bladder urothelial carcinoma and classification thereof
The specific process is as follows:
gDNA extraction
Bladder urothelial cancer cell lines were purchased from the cell bank of the Chinese academy of sciences. The extraction kit was purchased from QIAGEN (69506) and was performed according to the kit instructions.
DNA bisulfite conversion (reference example 2)
3. Biostandard gradient incorporation
RT4/5637gDNA treated by BS is subjected to ultrasonic disruption to simulate cfDNA, and single strand quantification is performed without ultrasonic disruption on normal urine cfDNA. By gradient incorporation according to the DNA concentration, 8 model standard product gradients (the gDNA content of the bladder urothelial carcinoma is 100%, 20%, 10%, 5%, 2%, 1%, 0.5%, 0%) are set in each group.
4. Methylation multiplex PCR, index PCR and 2 recoveries (reference example 2)
5. As a result, the
According to the data analysis results (see fig. 2), it can be seen that the invention can well detect tumors according to key areas. Wherein, in the cell line RT4 (non-muscle infiltrative), when the content of tumor DNA is more than 1 percent, the normal urine DNA sample and the sample containing the tumor DNA can be correctly distinguished by the method; in cell line 5637 (muscle layer infiltrates), tumor DNA content > 0.5% was well distinguishable from normal urine DNA samples, with good reproducibility 3 times.
Example 4
The present application incorporates 121 samples, including 27 tumor tissues from patients with urothelial carcinoma of the bladder, 70 preoperative urine from patients with urothelial carcinoma of the bladder, and 24 normal human urine.
The method carries out methylation multiplex PCR library construction on 121 samples to determine the judgment of distinguishing the non-muscle layer infiltrative property and the muscle layer infiltrative property of the method for the urinary epithelial cancer of the urinary tract of a normal person and a bladder, and comprises the following specific processes:
DNA extraction
The gDNA extraction kit was purchased from Meiji (D6315-03) or QIAGEN (69506) according to the kit instructions.
cfDNA extraction kits were purchased from Zymo (D3061) or Thermo Fisher (A29319) according to kit instructions.
Bisulfite conversion of DNA
Tissue gDNA was treated with BS at 200ng, cfDNA was treated with BS at 100ng (100 ng less was made up with. lamda. DNA purchased from Takala (3010). The specific procedure is as in example 2.
3. Methylation multiplex PCR, index PCR and 2 recoveries (reference example 2)
4. According to the multiple PCR off-line data, a generalized linear model is constructed for the methylation degree of each site, and bladder urothelial cancer tissues and urine are distinguished. From this model, other sample results are calculated.
5. Results
According to the results of the off-line data analysis (see fig. 3), the present method can distinguish the urothelial carcinoma of bladder from normal urine (AUC ═ 1.0) well, and in the urothelial carcinoma of bladder, the differentiation between the muscle layer wettability and the non-muscle layer wettability is also good (AUC ═ 0.969). In addition, in all urine samples, we can also find that the urine of patients with muscle layer infiltration and non-muscle layer infiltration (AUC 0.967) and the urine of tumor patients and normal people (AUC 0.94) have better discrimination.
The application aims at the detection sample which is urine cfDNA, the sensitivity and the specificity of the urine sample are calculated according to data, and specific results are shown in figure 3. When the application is used for diagnosing bladder urothelial cancer and normal people by using urine, the sensitivity is 0.889, and the specificity is 0.945; when urine was used to distinguish and classify the muscular-invasive bladder urothelial cancer and non-muscular-invasive bladder urothelial cancer, the sensitivity was 0.850 and the specificity was 1.000.
Sequence listing
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<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 5
tttccctaca cgacgctctt ccgatctnnn nnnnggtagg gttggaatga gaa 53
<210> 6
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 6
ttaatgcaac gatcgtcgaa attcgcnnnn nnnctctatt actaaaaccc caaaa 55
<210> 7
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 7
tttccctaca cgacgctctt ccgatctnnn nnnngtgagg gttggggatt atg 53
<210> 8
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 8
ttaatgcaac gatcgtcgaa attcgcnnnn nnncaaaact attctaccat aacaaac 57
<210> 9
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 9
tttccctaca cgacgctctt ccgatctnnn nnnntgaggg atttttaaaa gggg 54
<210> 10
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 10
ttaatgcaac gatcgtcgaa attcgcnnnn nnnaccctac caaaaaacta ttctt 55
<210> 11
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 11
tttccctaca cgacgctctt ccgatctnnn nnnnggttta gtttttagat attagaag 58
<210> 12
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 12
ttaatgcaac gatcgtcgaa attcgcnnnn nnnaaactaa ccctataacc cca 53
<210> 13
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 13
tttccctaca cgacgctctt ccgatctnnn nnnnttttat ataaaatttt tgtggatgg 59
<210> 14
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 14
ttaatgcaac gatcgtcgaa attcgcnnnn nnnacctaaa tactttaaaa ctaacct 57
<210> 15
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 15
tttccctaca cgacgctctt ccgatctnnn nnnngtttgg gtatttttat ttgtgaaa 58
<210> 16
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 16
ttaatgcaac gatcgtcgaa attcgcnnnn nnncaaaaac aaacacacaa actaac 56
<210> 17
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (42)..(42)
<223> y is c, t or u
<400> 17
tttccctaca cgacgctctt ccgatctnnn nnnnggggat tygtaataag tgg 53
<210> 18
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 18
ttaatgcaac gatcgtcgaa attcgcnnnn nnncraacaa acccctaaac tc 52
<210> 19
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 19
tttccctaca cgacgctctt ccgatctnnn nnnngtgtgg tttgagtgtt tgtt 54
<210> 20
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 20
ttaatgcaac gatcgtcgaa attcgcnnnn nnnaatcctt cactcaatcc cac 53
<210> 21
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 21
tttccctaca cgacgctctt ccgatctnnn nnnnggaaat tattttgggg attgag 56
<210> 22
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 22
ttaatgcaac gatcgtcgaa attcgcnnnn nnntaaacca aatatttcaa aaaaaacc 58
<210> 23
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 23
tttccctaca cgacgctctt ccgatctnnn nnnnaggaag gggttttagt taaag 55
<210> 24
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 24
ttaatgcaac gatcgtcgaa attcgcnnnn nnncctaaaa caaaaatcaa aatatcc 57
<210> 25
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 25
tttccctaca cgacgctctt ccgatctnnn nnnnggtagg agaaggttat ttatg 55
<210> 26
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 26
ttaatgcaac gatcgtcgaa attcgcnnnn nnncttttta aaatataaca cccatcc 57
<210> 27
<211> 56
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 27
tttccctaca cgacgctctt ccgatctnnn nnnngaggaa tttttttttg gtagga 56
<210> 28
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 28
ttaatgcaac gatcgtcgaa attcgcnnnn nnnccctaat tacaatacta acact 55
<210> 29
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 29
tttccctaca cgacgctctt ccgatctnnn nnnngatttg gtgaagagtt tttgg 55
<210> 30
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 30
ttaatgcaac gatcgtcgaa attcgcnnnn nnntaactat cacaccctac aatac 55
<210> 31
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (41)..(41)
<223> y is c, t or u
<400> 31
tttccctaca cgacgctctt ccgatctnnn nnnngttgag ygaggtttgg ag 52
<210> 32
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 32
ttaatgcaac gatcgtcgaa attcgcnnnn nnnaaatcta ctaaataaat acaacaca 58
<210> 33
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 33
tttccctaca cgacgctctt ccgatctnnn nnnntgttgg gaaagggagg g 51
<210> 34
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 34
ttaatgcaac gatcgtcgaa attcgcnnnn nnncccaact tcactttttc act 53
<210> 35
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 35
tttccctaca cgacgctctt ccgatctnnn nnnntggggt ttagagggat gg 52
<210> 36
<211> 55
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 36
ttaatgcaac gatcgtcgaa attcgcnnnn nnntctaata acccctacta atcac 55
<210> 37
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (34)..(34)
<223> n is a, c, g, t or u
<400> 37
tttccctaca cgacgctctt ccgatctnnn nnnngttagg gtttgtaggg ttt 53
<210> 38
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> primer sequences
<220>
<221> misc_feature
<222> (27)..(27)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (28)..(28)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (29)..(29)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (30)..(30)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (31)..(31)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (32)..(32)
<223> n is a, c, g, t or u
<220>
<221> misc_feature
<222> (33)..(33)
<223> n is a, c, g, t or u
<400> 38
ttaatgcaac gatcgtcgaa attcgcnnnn nnntaataaa actaaactac actaacctc 59

Claims (2)

1. Use of a primer combination for detecting a DNA methylation difference region in a test sample in the preparation of a reagent or kit for detecting or predicting whether a subject has urothelial bladder cancer or whether a subject is typed for muscle-invasive or non-muscle-invasive, wherein the DNA methylation difference region consists of regions in the genome of human hg19 version as shown below: chr 1: 86038429bp-86548655bp, chr 7: 23210805bp-23541085bp, chr 12: 50786446bp-51796719bp, chr 12: 53396863bp-55397136bp, chr 12: 51400340bp-55400617bp, chr 5: 2327659bp-2397867bp, chr 5: 5227879bp-5298195bp, chr 5: 14341021bp-14541266bp, chr 5: 15200260bp-15900461bp, chr 8: 2006233bp-2146598bp, chr 8: 102136659bp-102436833bp, chr 12: 130914602bp-130994836bp, chr 17: 77333510bp-77393712bp, chr 18: 3869388bp-3899760bp, chr 18: 11741967bp-11762330bp, chr 19: 1103012bp-1123479bp and chr 3: 180428100bp-181458540 bp; the primer combination consists of 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 and MN-DMR 4-1; the sequence of the forward primer in BLCBSP _ V1-4 is shown as SEQ ID NO. 1, and the sequence of the reverse primer in BLCBSP _ V1-4 is shown as SEQ ID NO. 2; the sequence of the forward primer in BLCBSP _ V1-9 is shown as SEQ ID NO. 3, and the sequence of the reverse primer in BLCBSP _ V1-9 is shown as SEQ ID NO. 4; the forward primer sequence in BLCBSP _ V1-13 is shown as SEQ ID NO. 5, and the reverse primer sequence in BLCBSP _ V1-13 is shown as SEQ ID NO. 6; the forward primer sequence in BLCBSP _ V1-14 is shown as SEQ ID NO. 7, and the reverse primer sequence in BLCBSP _ V1-14 is shown as SEQ ID NO. 8; the forward primer sequence in BLCBSP _ V1-15 is shown as SEQ ID NO. 9, and the reverse primer sequence in BLCBSP _ V1-15 is shown as SEQ ID NO. 10; the sequence of the forward primer in BLCBSP _ V2-7 is shown as SEQ ID NO. 11, and the sequence of the reverse primer in BLCBSP _ V2-7 is shown as SEQ ID NO. 12; the sequence of the forward primer in BLCBSP _ V2-8 is shown as SEQ ID NO. 13, and the sequence of the reverse primer in BLCBSP _ V2-8 is shown as SEQ ID NO. 14; the forward primer sequence in BLCBSP _ V2-10 is shown as SEQ ID NO. 15, and the reverse primer sequence in BLCBSP _ V2-10 is shown as SEQ ID NO. 16; the sequence of the forward primer in BLCBSP _ V2-11 is shown as SEQ ID NO. 17, and the sequence of the reverse primer in BLCBSP _ V2-11 is shown as SEQ ID NO. 18; the forward primer sequence in BLCBSP _ V2-16 is shown as SEQ ID NO. 19, and the reverse primer sequence in BLCBSP _ V2-16 is shown as SEQ ID NO. 20; the forward primer sequence in BLCBSP _ V2-17 is shown as SEQ ID NO:21, and the reverse primer sequence in BLCBSP _ V2-17 is shown as SEQ ID NO: 22; the forward primer sequence in BLCBSP _ V2-18 is shown as SEQ ID NO. 23, and the reverse primer sequence in BLCBSP _ V2-18 is shown as SEQ ID NO. 24; the forward primer sequence in BLCBSP _ V2-19 is shown as SEQ ID NO. 25, and the reverse primer sequence in BLCBSP _ V2-19 is shown as SEQ ID NO. 26; the forward primer sequence in BLCBSP _ V2-21 is shown as SEQ ID NO. 27, and the reverse primer sequence in BLCBSP _ V2-21 is shown as SEQ ID NO. 28; the forward primer sequence in BLCBSP _ V2-22 is shown as SEQ ID NO. 29, and the reverse primer sequence in BLCBSP _ V2-22 is shown as SEQ ID NO. 30; the forward primer sequence in BLCBSP _ V2-24 is shown as SEQ ID NO. 31, and the reverse primer sequence in BLCBSP _ V2-24 is shown as SEQ ID NO. 32; the forward primer sequence in BLCBSP _ V2-33 is shown as SEQ ID NO. 33, and the reverse primer sequence in BLCBSP _ V2-33 is shown as SEQ ID NO. 34; the forward primer sequence in BLCBSP _ V2-35 is shown as SEQ ID NO. 35, and the reverse primer sequence in BLCBSP _ V2-35 is shown as SEQ ID NO. 36; the forward primer sequence in MN-DMR4-1 is shown as SEQ ID NO:37, and the reverse primer sequence in MN-DMR4-1 is shown as SEQ ID NO: 38.
2. The use according to claim 1, wherein the sample to be tested is urine cfDNA or bladder urothelial cancer tissue gDNA derived from the subject.
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