CN116814781A - Markers, kits and devices for detecting urothelial cancer - Google Patents

Abstract

The present invention relates to markers for detecting urothelial cancer, including CpG islands of ZNF551 and/or CYTH2 genes. The markers disclosed in the present invention for detecting urothelial cancer include CpG islands of ZNF551 and/or CYTH2 genes. According to the methylation level of the CpG island of the target gene of the detection sample, urothelial cancer samples and healthy samples can be distinguished. While achieving non-invasive detection, it also improves the sensitivity and specificity of urothelial cancer detection.

Description

Markers, kits and devices for detecting urothelial cancer

This application is a divisional application of an invention patent application with a filing date of December 23, 2021, an application number of 202111588302.1, and an invention name of “Markers, kits and devices for detecting urothelial cancer”.

Technical field

The present invention relates to the field of cancer, and in particular, to a marker, a kit and a device for detecting urothelial cancer.

Background technique

Urothelial cancer is a tumor originating from the epithelial lining of the kidneys, ureters, bladder and urethra. It mainly includes upper urinary tract urothelial cancer (ureteral cancer and renal pelvis cancer) and bladder cancer, of which the former accounts for the highest incidence rate. 5%-10%, the incidence of the latter accounts for 90%-95%. The incidence rate of bladder cancer is three times that of the mortality rate. It is a cancer with a high cure rate. About 70% of patients relapse after transurethral resection. Early detection of bladder cancer can increase the chance of surgical preservation of the bladder and improve the overall survival of the patient. Early detection and early diagnosis are the keys to improving the cure rate of bladder cancer.

Currently, a variety of methods have been developed for the detection and diagnosis of bladder cancer. Cystoscopy tissue biopsy is the gold standard for bladder cancer diagnosis. However, because it is an invasive and invasive test, patient acceptance is low.

Urine cytology examination of urine or bladder wash specimens is one of the main methods for bladder cancer diagnosis and postoperative follow-up. The sensitivity of urine exfoliated cytology examination is about 13%-75%, and the specificity is about 85%-100 %.

Due to the low sensitivity of urine cytology, a variety of urine bladder tumor marker detection technologies have been developed. The bladder cancer urine marker detection methods approved by the US FDA include nuclear matrix protein 22 (NMP22), bladder tumor antigen (BTA) ), immune-cell examination (ImmunoCyt), fibrinogen degradation products (FB/FDP) and fluorescence in situ hybridization (FISH), etc. Others include: telomerase, survivin, microsatellite analysis, cytokeratin examination, etc. The above tumor markers have shown high sensitivity in clinical studies for detecting bladder cancer, but the specificity is lower than that of urine cytology. .

Therefore, it is difficult to ensure the sensitivity and high specificity of non-invasive detection of bladder cancer at the same time.

Contents of the invention

In order to solve the above problems, the first object of the present invention is to provide a marker for detecting urothelial cancer, including the CpG island of at least one gene of NBR1, ZNF551, CYTH2 and ARL5C, to solve the problem of existing non-invasive bladder cancer detection methods Problems with low sensitivity or low specificity.

In one implementation of the present invention, taking GRCh38.p13 as the reference genome, the CpG island of the NBR1 gene includes the sequence of the Chr17:43211709-43212254 region, the CpG island of the ZNF551 gene includes the sequence of the Chr19:57708822-57709149 region, and the CpG island of the CYTH2 gene The CpG island includes the sequence of the Chr19:48480306-48480918 region, and the CpG island of the ARL5C gene includes the sequence of the Chr17:39165230-39165846 region.

In one implementation of the present invention, the CpG island of the NBR1 gene includes the positive strand of the Chr17:43211712-43211925 region, the positive strand of the Chr17:43212002-43212173 region, the negative strand of the Chr17:43212230-43212079 region and Chr17:43212042-43211875 The negative strand sequence of the region;

The CpG island of the ZNF551 gene includes the positive strand sequence of the Chr19:57708924-57709057 region and the negative strand sequence of the Chr19:57709100-57708959 region;

The CpG island of the CYTH2 gene includes the positive strand of the Chr19:48480372-48480527 region, the positive strand of the Chr19:48480517-48480664 region, the negative strand of the Chr19:48480899-48480743 region, and the negative strand sequence of the Chr19:48480624-48480446 region;

The CpG island of the ARL5C gene includes the positive strand of the Chr17:39165294-39165443 region, the positive strand of the Chr17:39165465-39165635 region, the negative strand of the Chr17:39165768-39165591 region, and the negative strand sequence of the Chr17:39165534-39165295 region.

The second object of the present invention is to provide the use of a reagent for detecting the methylation level of the above marker in preparing a kit for detecting urothelial cancer.

The third object of the present invention is to provide a kit for detecting urothelial cancer, including a reagent for detecting the methylation level of the above marker.

In one implementation of the present invention, the reagent includes at least one of a primer pair and a probe for detecting the methylation level of the marker.

In one implementation of the present invention, the primer pair includes SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.5, and SEQ ID NO.7 for amplifying the CpG island of the NBR1 gene. and SEQ ID NO.8, SEQ ID NO.10 and SEQ ID NO.11, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.16 and SEQ ID NO. used to amplify the CpG island of the ZNF551 gene. 17. SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.22 and SEQ ID NO.23, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO. used to amplify the CpG island of CYTH2 gene. 28 and SEQ ID NO. 29, and SEQ ID NO. 31 and SEQ ID NO. 32, SEQ ID NO. 34 and SEQ ID NO. 35, SEQ ID NO. 37 and SEQ ID NO. 37 for respectively amplifying the CpG island of the ARL5C gene. NO.38, at least one set of sequences in SEQ ID NO.40 and SEQ ID NO.41.

In one implementation of the present invention, the probe includes SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, and SEQ ID NO.12 for detecting NBR1 gene CpG island, and is used for detecting ZNF551 gene CpG island. SEQ ID NO.15, SEQ ID NO.18, SEQ ID NO.21, SEQ ID NO.24, SEQ ID NO.27, SEQ ID NO.30 for detecting CYTH2 gene CpG island, and SEQ ID NO.21 for detecting ARL5C gene CpG At least one sequence of SEQ ID NO.33, SEQ ID NO.36, SEQ ID NO.39, and SEQ ID NO.42 of the island.

In one implementation of the present invention, the kit further includes at least one of a nucleic acid extraction reagent, a nucleic acid conversion reagent, a PCR reaction reagent, a positive control sample and a negative control sample.

In one implementation of the present invention, urothelial cancer includes at least one of renal pelvis cancer, ureteral cancer and bladder cancer.

The fourth object of the present invention is to provide a device for detecting urothelial cancer, including:

Methylation information acquisition module: used to obtain the methylation information of the CpG island of at least one gene of NBR1, ZNF551, CYTH2 and ARL5C in the sample to be tested, where the sample to be tested is a DNA sample after nucleic acid conversion;

Sample type judgment module: used to judge whether the sample type to be tested is a urothelial cancer sample or a healthy sample based on the methylation information.

The present invention also relates to a computer-readable storage medium. The computer storage medium is used to store computer instructions, programs, code sets or instruction sets, which when run on a computer, cause the computer to execute to implement the above-mentioned detection of urothelial cancer. All steps include:

Obtain the methylation information of the CpG island of at least one gene of NBR1, ZNF551, CYTH2 and ARL5C in the sample to be tested, where the sample to be tested is a DNA sample after nucleic acid conversion;

The type of sample to be tested is determined to be a urothelial cancer sample or a healthy sample based on the methylation information.

The invention also relates to an electronic device, including:

one or more processors; and

a storage device that stores one or more programs,

When one or more programs are executed by one or more processors, the one or more processors implement all the above steps for detecting urothelial cancer. All steps include:

Obtain the methylation information of the CpG island of at least one gene of NBR1, ZNF551, CYTH2 and ARL5C in the sample to be tested, where the sample to be tested is a DNA sample after nucleic acid conversion;

The type of sample to be tested is determined to be a urothelial cancer sample or a healthy sample based on the methylation information.

The markers disclosed in the present invention for detecting urothelial cancer include the CpG island of at least one gene of NBR1, ZNF551, CYTH2 and ARL5C. According to the methylation of the CpG island of at least one gene of NBR1, ZNF551, CYTH2 and ARL5C in the detection sample Information can distinguish urothelial cancer samples from healthy samples, while achieving non-invasive detection and improving the sensitivity and specificity of urothelial cancer detection.

Description of the drawings

In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings that need to be used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description The drawings illustrate some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting any creative effort.

Figure 1 is a schematic diagram of ROC curve analysis of methylation levels of marker regions 1 to 14 in Example 1 of the present invention.

Detailed ways

Reference is now provided in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment, to yield still further embodiments.

It is therefore intended that the present invention cover the modifications and variations described above as falling within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the invention are disclosed in, or are apparent from, the following detailed description. Those of ordinary skill in the art will appreciate that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the invention.

The term "marker" or "biochemical marker" as used herein refers to the methylation level of a target molecule to be used in the analysis of a patient experimental sample. Examples of such molecular targets are nucleic acids, proteins or polypeptides.

As used herein, the term "primer" refers to an oligonucleotide, whether naturally occurring in a purified restriction digest or produced synthetically, which oligonucleotide is positioned to induce the synthesis of a primer extension product complementary to the nucleic acid strand. Oligonucleotides can function as starting points for synthesis when provided under conditions (eg, in the presence of a nucleotide and an inducer such as a DNA polymerase and at appropriate temperature and pH). Primers are preferably single-stranded for maximum efficiency in amplification, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primers are oligodeoxyribonucleotides. The primer should be long enough to trigger the synthesis of the extension product in the presence of the inducer. The exact length of the primer will depend on many factors, including temperature, primer source, and method used. For example, in some embodiments, primers range from 10-100 or more nucleotides (e.g., 10-300, 15-250, 15-200, 15-150, 15-100, 15-90, 20-80 , 20-70, 20-60, 20-50 nucleotides, etc.).

In the present invention, the tissue or cancer may be from a mammal and is preferably from a human, although monkey, ape, cat, dog, cow, horse and rabbit are also within the scope of the invention.

The "CpG island" referred to in the present invention is the same as the general understanding. CpG is the abbreviation of cytosine-phosphate-guanine. CpG islands are some regions rich in CpG dinucleotides, with a length of 300-3000 bp and a GC content greater than 50%. , CpG islands are mainly located in the promoter and exon regions of genes, and may also be located in other regions such as UTR and intron regions. In actual operations, software, programs or manual methods can be used to set parameters and find CpG islands in genes. CpG Island.

The term "methylation level" is the same as generally understood, and refers to whether the cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency/ratio/percentage of methylation, It represents both qualitative and quantitative concepts. For example, if a cytosine (C) residue within a nucleic acid sequence is methylated, it can be said to be "hypermethylated" or have "increased methylation." In practical applications, this can be determined based on the actual Different detection indicators are used to compare DNA methylation levels. For example, in some cases, the comparison can be based on the Ct value of the sample fluorescence quantitative PCR detection. In some cases, the methylation ratio of the marker in the sample can be calculated. That is, the number of methylated molecules/(the number of methylated molecules + the number of non-methylated molecules) × 100, and then comparison is made. In some cases, statistical analysis and integration of various indicators are required to obtain the final judgment index.

The "methylation level of a CpG island" referred to in the present invention refers to the methylation level of a nucleic acid sequence containing at least one CpG dinucleotide site within a CpG island. The first object of the present invention is to provide a marker for detecting urothelial cancer, including the CpG island of at least one gene of NBR1, ZNF551, CYTH2 and ARL5C, to solve the problem of low sensitivity or specificity of existing non-invasive bladder cancer detection methods. low question.

In some embodiments, taking GRCh38.p13 as the reference genome, the CpG island of the NBR1 gene includes the sequence of the Chr17:43211709-43212254 region, the CpG island of the ZNF551 gene includes the sequence of the Chr19:57708822-57709149 region, and the CpG island of the CYTH2 gene includes The sequence of the Chr19:48480306-48480918 region, and the CpG island of the ARL5C gene includes the sequence of the Chr17:39165230-39165846 region.

In some embodiments, the CPG Island of the NBR1 gene includes the positive chain of the CHR17: 43211712-43211925 area, CHR17: 43212002-43212173, CHR17: 4321223212079's negative chain and CHR17: 43212042-4. The negative of the 3211875 area chain sequence;

The CpG island of the ZNF551 gene includes the positive strand sequence of the Chr19:57708924-57709057 region and the negative strand sequence of the Chr19:57709100-57708959 region;

The CpG island of the CYTH2 gene includes the positive strand of the Chr19:48480372-48480527 region, the positive strand of the Chr19:48480517-48480664 region, the negative strand of the Chr19:48480899-48480743 region, and the negative strand sequence of the Chr19:48480624-48480446 region;

The CpG island of the ARL5C gene includes the positive strand of the Chr17:39165294-39165443 region, the positive strand of the Chr17:39165465-39165635 region, the negative strand of the Chr17:39165768-39165591 region, and the negative strand sequence of the Chr17:39165534-39165295 region.

A second aspect of the present invention provides the use of a reagent for detecting the methylation level of the above marker in preparing a kit for detecting urothelial cancer.

A third aspect of the present invention provides a kit for detecting urothelial cancer, including a reagent for detecting the methylation level of the above marker.

In some embodiments, the reagents include at least one of a primer pair and a probe for detecting the marker.

In some embodiments, the primer pair includes SEQ ID NO. 1 and SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO. for amplifying the CpG island of the NBR1 gene. .8, SEQ ID NO.10 and SEQ ID NO.11, SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.16 and SEQ ID NO.17, used for amplification of CpG island of ZNF551 gene, used for amplification SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.22 and SEQ ID NO.23, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.28 and SEQ ID NO. of the CYTH2 gene CpG island. 29, and SEQ ID NO.31 and SEQ ID NO.32, SEQ ID NO.34 and SEQ ID NO.35, SEQ ID NO.37 and SEQ ID NO.38, SEQ ID used to amplify the ARL5C gene CpG island respectively. NO.40 and at least one set of sequences in SEQ ID NO.41.

In some embodiments, the probes include SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12 for detecting CpG island of NBR1 gene, SEQ ID NO.12 for detecting CpG island of ZNF551 gene. NO.15, SEQ ID NO.18, SEQ ID NO.21, SEQ ID NO.24, SEQ ID NO.27, SEQ ID NO.30 for detecting CpG island of CYTH2 gene, and SEQ for detecting CpG island of ARL5C gene At least one sequence among ID NO.33, SEQ ID NO.36, SEQ ID NO.39, and SEQ ID NO.42.

In some embodiments, the kit further includes at least one of a nucleic acid extraction reagent, a nucleic acid conversion reagent, a PCR reaction reagent, a positive control sample, and a negative control sample.

In some embodiments, urothelial cancer includes at least one of renal pelvis cancer, ureteral cancer, and bladder cancer.

A fourth aspect of the present invention also provides a method for detecting urothelial cancer, including:

Obtain the methylation information of the CpG island of at least one gene of NBR1, ZNF551, CYTH2 and ARL5C in the sample to be tested, where the sample to be tested is a DNA sample after nucleic acid conversion;

The type of sample to be tested is determined to be a urothelial cancer sample or a healthy sample based on the methylation information.

Specifically, the samples to be tested include at least one of urine samples, bladder lavage samples, and blood samples. Nucleic acid conversion processing is a common step for determining DNA methylation levels. It refers to using nucleic acid conversion reagents to process sample DNA and convert the sample into The cytosine residues in the DNA are converted into uracil, while the originally methylated cytosine residues in the sample DNA are not affected. The nucleic acid conversion reagents used include but are not limited to bisulfite.

In some specific embodiments, the methylation information of the CpG island refers to the methylation level of the CpG island, and the methylation level of the CpG island of each gene is obtained by at least one of the following methods: methylation specificity PCR method, sequencing method, methylation-specific high-performance liquid chromatography method, digital PCR method, methylation-specific high-resolution melting curve method, methylation-specific microarray method, methylation-sensitive restriction Endonuclease method and flap endonuclease method.

Furthermore, based on the methylation information of urothelial cancer samples and healthy samples, a classification model was constructed to determine the sample type based on the ROC curve analysis results, and the cutoff value for distinguishing urothelial cancer samples and healthy samples was determined, and then based on the methylation information of the samples to be tested. Whether the methylation level exceeds the cut-off value can determine whether the sample to be tested is a urothelial cancer sample or a healthy sample. For a sample to be tested with a methylation level less than or equal to the cut-off value, it can be determined to be a urothelial cancer sample. For methylation Samples to be tested with levels greater than the cutoff value can be identified as healthy samples.

A fifth aspect of the present invention also provides a device for detecting urothelial cancer, including:

Methylation information acquisition module: used to obtain the methylation information of the CpG island of at least one gene of NBR1, ZNF551, CYTH2 and ARL5C in the sample to be tested, where the sample to be tested is a DNA sample after nucleic acid conversion;

Sample type judgment module: used to judge whether the sample type to be tested is a urothelial cancer sample or a healthy sample based on the methylation information.

The present invention also relates to a computer-readable storage medium. The computer storage medium is used to store computer instructions, programs, code sets or instruction sets, which when run on a computer, cause the computer to perform all the steps of predicting urothelial cancer. , all steps include:

Obtain the methylation information of the CpG island of at least one gene of NBR1, ZNF551, CYTH2 and ARL5C in the sample to be tested, where the sample to be tested is a DNA sample after nucleic acid conversion;

The type of sample to be tested is determined to be a urothelial cancer sample or a healthy sample based on the methylation information.

A computer-readable signal medium may include a data signal propagated in baseband or as part of a carrier wave carrying computer-readable program code therein. Such propagated data signals may take many forms, including but not limited to electromagnetic signals, optical signals, or any suitable combination of the above. A computer-readable signal medium may also be any computer-readable medium other than a computer-readable storage medium that can send, propagate, or transmit a program for use by or in connection with an instruction execution system, apparatus, or device . Program code embodied on a computer-readable medium may be transmitted using any appropriate medium, including but not limited to wireless, wire, optical cable, RF, etc., or any suitable combination of the foregoing.

Computer program code for performing operations of the present disclosure may be written in one or more programming languages, including object-oriented programming languages such as Java, Smalltalk, C++, and conventional procedural programming languages, or a combination thereof. Programming language - such as "C" or a similar programming language. Program code may execute entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly remotely computer, or execute entirely on a remote computer or server. In the case of a remote computer, the remote computer can be connected to the user's computer through any kind of network—including a local area network (LAN) or a wide area network (WAN)—or it can Connect to an external computer (such as using an Internet service provider to connect via the Internet).

The invention also relates to an electronic device, including:

one or more processors; and

a storage device that stores one or more programs,

When one or more programs are executed by one or more processors, the one or more processors implement all the above steps for detecting urothelial cancer. All steps include:

Obtain the methylation information of the CpG island of at least one gene of NBR1, ZNF551, CYTH2 and ARL5C in the sample to be tested, where the sample to be tested is a DNA sample after nucleic acid conversion;

The type of sample to be tested is determined to be a urothelial cancer sample or a healthy sample based on the methylation information.

Optionally, the electronic device may also include a transceiver. The processor and transceiver are connected, for example via a bus. It should be noted that the number of transceivers in practical applications is not limited to one, and the structure of the electronic device does not constitute a limitation on the embodiments of the present application.

The processor may be a CPU, a general-purpose processor, a DSP, an ASIC, an FPGA or other programmable logic devices, transistor logic devices, hardware components or any combination thereof. It may implement or execute the various illustrative logical blocks, modules, and circuits described in connection with this disclosure. A processor can also be a combination that implements computing functions, such as a combination of one or more microprocessors, a combination of a DSP and a microprocessor, etc.

A bus may include a path that carries information between the above-mentioned components. The bus can be PCI bus or EISA bus, etc. The bus can be divided into address bus, data bus, control bus, etc. Memory 802 can be ROM or other types of static storage devices that can store static information and instructions, RAM or other types of dynamic storage devices that can store information and instructions, or it can be EEPROM, CD-ROM or other optical disk storage, optical disk storage (including compressed optical discs, laser discs, optical discs, digital versatile discs, Blu-ray discs, etc.), disk storage media or other magnetic storage devices, or can be used to carry or store desired program code in the form of instructions or data structures and can be used by a computer Any other medium for access, but not limited to this.

To be clear, the ideal scenario for diagnosis is one in which a single event or process causes various diseases, for example, in infectious diseases. In all other cases, correct diagnosis can be very difficult, especially when the etiology of the disease is not fully understood, as is the case with many cancer types. As the skilled artisan will appreciate, no biochemical marker for diagnosis of a given multifactorial disease is 100% specific and 100% sensitive. Conversely, biochemical markers (eg, CpG islands of at least one gene NBR1, ZNF551, CYTH2, and ARL5C) can be used to assess, for example, the presence or absence of a disease with some probability or predictive value. Therefore, in routine clinical diagnosis, various clinical symptoms and biological markers are usually comprehensively considered to diagnose, treat, and control underlying diseases.

The embodiments of the present invention will be described in detail below with reference to examples.

Example 1

1. Sample collection and processing

The inclusion criteria for patients with urothelial cancer were: urinary exfoliated cytology, or urinary tract CT plain enhanced scan, or retrograde upper urography, or diagnostic ureterocystoscopy, and pathologically confirmed urothelial cancer. crowd.

The inclusion criteria for the normal population are: those whose cystoscopy report shows no space-occupying lesions, or those whose urinary system ultrasound examination reports no abnormality in the bladder and three consecutive urine exfoliated cytology reports show no cancer cells.

Collect urine samples from urothelial cancer patients and normal people, collect 15 mL from each person, add EDTA to preserve, the final concentration of EDTA is 40mM.

2. Genome extraction

Before genome extraction, mix the urine sample thoroughly and take 2mL of urine for genome extraction. The reagents used for extraction are nucleic acid extraction reagents from Wuhan Amison Life Technology Co., Ltd. (model: rapid extraction and conversion of free DNA). The specific operations are as follows:

a. Cleavage binding: Prepare a clean 5ml centrifuge tube and add 100μL proteinase K to it. Mix the urine and distribute 2 mL into the tube containing proteinase K. Add 2 ml of cleavage binding solution and 20 μL of magnetic beads in sequence. After mixing by inverting, place it on a square mixer for lysis at room temperature for 30 minutes, keeping the magnetic beads in a suspended state. .

b. Washing: Place the centrifuge tube on the magnetic stand and absorb the magnet for 2 minutes. After the solution is clear, invert the tube several times to rinse the remaining magnetic beads on the cap until all the magnetic beads are absorbed. Carefully absorb the waste liquid, add 2 mL of washing solution, vortex and mix more than 10 times to completely disperse the magnetic beads, and absorb the magnet for 2 minutes. After the solution is clear, invert the tube several times and cover the remaining magnetic beads until all the magnetic beads are absorbed. Finish.

c. Rinse: Carefully absorb the waste liquid, first add 500μL rinse solution to wash the magnetic beads to the bottom, then transfer the magnetic bead suspension to a new 2.0mL centrifuge tube, then add 500μL rinse solution to remove the residue on the wall of the 5mL centrifuge tube. Completely wash the magnetic beads to the bottom, centrifuge briefly and transfer all the magnetic bead suspension to a 2.0 mL centrifuge tube, vortex and mix more than 10 times to completely disperse the magnetic beads, absorb magnet for 2 minutes, until the solution is clear, invert and rinse several times Cover the tube with remaining magnetic beads until all magnetic beads are absorbed. Repeat rinsing once.

d. Elution: Remove the centrifuge tube and centrifuge briefly to collect the remaining liquid. Place it on a magnetic stand. After the magnetic absorption is completed, use a small pipette tip to absorb the remaining liquid. Open the centrifuge tube and leave it for 5 minutes to make the surface of the magnetic beads matte. Add 50 μL eluent TE, shake gently to disperse the magnetic beads, place it at 56°C for elution for 10 minutes, take it out every 3 minutes and shake gently to keep the magnetic beads in a suspended state.

e. Collection: Take out the centrifuge tube, centrifuge to collect the liquid on the tube cover and tube wall, place it on a magnetic stand to attract magnets for 1 minute, and carefully absorb the supernatant DNA solution.

3. Conversion of sulfite

The extracted genome was subjected to bisulfite conversion. The nucleic acid conversion kit used was the nucleic acid conversion reagent of Wuhan Amison Life Technology Co., Ltd. (Ehan Mechanical Preparation 20200843). For specific experimental operations, please refer to the kit instructions. In this process, unmethylated cytosine (C) is converted into uracil (U), methylated cytosine remains unchanged, and uracil is paired with adenine (A) in the subsequent PCR step. The converted cytosine pairs with guanine (G) to distinguish between methylated and unmethylated sequences.

4. PCR reaction

The methylation-specific PCR method was used to detect 4 regions on the NBR1 gene, 2 regions on the ZNF551 gene, 4 regions on the CYTH2 gene, 4 regions on the ARL5C gene, and two regions on the POU4F2 gene, TWIST1 The methylation levels of CpG islands in two regions on the gene, one region on the VIM gene and one region on the ZNF154 gene were detected. The positions of each region on the chromosome, primers and probes for PCR amplification are shown in Table 1.

Table 1

In Table 1, regions 1, 2, 5, 7, 8, 11, 12, 15, 16, 17, 18, 19 and 20 are located on the positive strand of the genome, and regions 3, 4, 6, 9, 10, 13 and 14 are located on the positive strand of the genome. On the negative strand of the genome.

The probe that detects the target region is a Taqman probe, the reporter group at the 5' end is FAM, and the quencher group at the 3' end is MGB.

(2) Preparation of positive control and negative control

During PCR amplification, the positive and negative controls in regions 1-20 are synthetic sequences constructed on the vector. The base composition of the synthetic sequences is designed with reference to the sequences of the CpG regions of the four genes. All cells in the negative control The C positions of the pyrimidine are all designed as T. In the positive control, except for the C position of the CG dinucleotide, all the C positions are designed as T. The nucleotides at other positions have the same sequence as the CpG island region.

The synthetic sequence of the positive control in regions 1-4 is shown in SEQ ID NO. 61 (5’-3’):

CGGAAGTAGCGTTCGGGTTTCGGTAACGTTTGAAGAGTTGGTAGCGTTTTAGGTTGTTGCGTGGCGAAGGGGCCGGATCGGGGGACGGGGGGGTGGGTTTTTAGGGGTTGGGGCGGGATTTTTTTGGGTATTGAGTTAAAGTTTGAGGGGGAGTGTTCGTTTTCGTATTTTCGATTTTTTTTTTCGTTTTTGTCGTTGTAGTCGTTCGCGGGTAAGGTTTTTTTTTAGATTTTTTATTATTATT TTATTTTTCGGAGGTTAAGATATTTTTTGTTGTCGTTTTTTCGTTTAATATATAGGCGTTTTTCGTACGGGTATTAAGTTTGATTAACGGTTTTTAGGGGTGTGGTGGGATTTGAAATTTTTTTTTAATGAAGTTATATTTTTTTCGTTCGTTTTTATTCGGTCGGTTTATGGTAGAGTTTTTATGTAGTTTAGGAATCGGTTTGGTTTTTCGGGGAGGTTAGCGTTTTTATTGTTTTTATTTTTTAGTGCG GTATATTCGTTTTTTTAGGATTTCGGTCGGTTGTCGGTTTTAGGCG (SEQ ID NO. 61).

The synthetic sequence of the negative control in regions 1-4 is shown in SEQ ID NO. 62 (5’-3’):

TGGAAGTAGTGTTTGGGTTTTTGGGTAATGTTTGAAGAGTTGGTAGTGTTTTAGGTTGTTGTGTGGTGAAGGGGTGGATTGGGGGATGGGGGGGTGGGTTTTTAGGGGTTGGGGTGGGATTTTTTTGGGTATTGAGTTAAAGTTTGAGGGGGAGTGTTTGTTTTTGTATTTTTGATTTTATTTTTTTTTGTTTTTGTTGTTGTAGTTGTTTGTGGGTAAGGTTTTTTTTTAGATTTTTTTTTTTTTT ATTTTTTGGAGGTTAAGATATTTTTTGTGTTGTTTTTTTGTTTAATATATAGGTGTTTTTTGTATGGGTATTAAGTTTGATTAATGGTTTTTAGGGGTGTGGTGGGATTTGAAATTTTTTTTTAATGAAGTTATATTTTTTTTGTTTGTTTTTATTTGGTTGGTTTATGGTAGAGTTTTTATGTAGTTTAGGAATTGGTTTGGTTTTTTGGGGAGGTTAGGTGTTTTTATTGTTTTTATTTTAGTGTGGT ATATTTGTTTTTTTAGGATTTTGGTTGGTTGTTGGTTTTAGGTG (SEQ ID NO. 62).

The synthetic sequence of the positive control in region 5-6 is shown in SEQ ID NO. 63 (5’-3’):

CGAACGTTTTAGTGTTTTCGATTTTGGGTAGCGGGGATTCGAGTAGGCGTTTTTTATTGATGGTTTTAGAACGTGGGTGGGGGAAGGTGTGTGAGGACGGGAAGACGTCGTATTTATTTGAGTTGGCGTTTTTAGAGTGGTCGTTGTTATTAGATTTTGCGGGTAGAGTTGGGTCGGGAGCGACGGGCGATATTGGTAGGGATTCGGGGATAGCGGTTTTTTTTAGGTTTGACGTGGGTTTTTTTT AGGGCGGCGTCGTTAAGGTTTAGACGTTTTCGTGTAGGAGGGACGACGATTTTTTTTACGTTTTCGTGGTTTTTAATTCGGCG (SEQ ID NO. 63).

The synthetic sequence of the negative control in region 5-6 is shown in SEQ ID NO. 64 (5’-3’):

TGAATGTTTTAGTGTTTTTGATTTTGGGTAGTGGGGATTTGAGTAGGTGTTTTTTATTGATGGTTTTAGAATGTGGGTGGGGGAAGGTGTGTGAGGATGGGAAGATGTTGTATTTATTTGAGTTGGTGTTTTTAGAGTGGTTGTTGTTATTAGATTTTGTGGGTAGAGTTGGGTTGGGAGTGATGGGTGATATTGGTAGGGATTTGGGGATAGTGGTTTTTTTTAGGTTTGATGTGGGTTTTTTTT AGGGTGGTGTTGTTAAGGTTTAGATGTTTTTGTGTAGGAGGGATGATGATTTTTTTTATGTTTTTGTGGTTTAATTTGGTG (SEQ ID NO. 64).

The synthetic sequence of the positive control in regions 7-10 is shown in SEQ ID NO. 65 (5’-3’):

CGGGACGCGGGTGGGAGGCGTTTGTGGTTTCGAGGCGTGTCGCGAGTTGTAGTTTTTTTTGTTCGTTGTGTTCGTTTTTGAGTTTTTCGATGGGATGCGGCGTTTCGGAATTTCGGGTTTTGATTTTTGTTTCGTTTTTGGTTATAGGTATTTGTCGGTTTGAAGGTTTCCGGTGGGGGTATTTTGCGTTTTTTCGCGGGAAGGTGGATTATAGTTATCGGTAGGTTGTGCGGCGTTAAAGTTAC GGTGATTTAGATTCGAGGTTTTTTCGGGCGTCGTAGTTTTTCGAGATTTCGTGGCGGCGTTTGTTTTTTTTTTTAGTTAGATTTCGTATTTTTGTGGAGGGTAGAGGAGGTTTTGATCGTCGCGGTTTCGGGGGTTGGTGGGAAATGGAGTTTTAAATGAGAAAATAGAATTTTTTATTTTTTTTTTTTATAGGTGTTAGGGAAACGGAGTTATCGATTTAAGAAGGTCGTGGGAGATGAGGTTTTAGGGTAAATA GCGGGTTTCGTTATTATGTTATTTTTTTTTGTCGTTTTTTCGGATGAATTGTATGTAGGGCGGTCGGTTTCGTGGTAGGTAGAGGTAGGAAGAGGCGCGGAGTTCG (SEQ ID NO. 65).

The synthetic sequence of the negative control in regions 7-10 is shown in SEQ ID NO. 66 (5’-3’):

TGGGATGTGGGTGGGAGGTGTTTGTGGTTTTGAGGTGTGTTGTGAGTTGTAGTTTTTTTTGTTTGTTGTGTTTGTTTTTGAGTTTTTTGATGGGATGTGGTGTTTTGGAATTTTGGGTTTTGATTTTTGTTTTGTTTTTGGTTATAGGTATTTGTTGGTTTGAAGGTTTTTGTGGTGGGGGTATTTTGTGTTTTTTTGTGGGAAGGTGGATTATAGTTATTGGTAGGTTGTGTGGTGTTAAAGTTAT GGTGATTTAGATTTGAGGTTTTTGGGTGTTGTAGTTTTTTGAGATTTTGTGGTGGTGTTTGTTTTTTTTTAGTTAGATTTTGTATTTTTGTGGAGGGTAGAGGAGGTTTTGATTGTTGTGGTTTTGGGGGTTGGTGGGAAATGGAGTTTTAAATGAGAAAATAGAATTTTTTATTTTTTTTTTTTATAGGTGTTAGGGAAATGGAGTTATTGATTTAAGAAGGTTGTGGGAGATGAGGTTTTAGGGTAAATA GTGGGTTTTGTTATTATGTTATTTTTTTTTGTTGTTTTTTTGGATGAATTGTATGTAGGGTGGTTGGTTTTGTGGTAGGTAGAGGTAGGAAGAGGTGTGGAGTTTG (SEQ ID NO. 66).

The synthetic sequence of the positive control in region 11-14 is shown in SEQ ID NO. 67 (5’-3’):

CGTTTTTTCGTAGGTTGGGGATTTTTGTTTATCGTATCGCGTTTGTTTTTTATTTAGTTTCGATATTTGTTTTATTTCGGGACGGTGGAAAAGATAGTTGAGTTTTTATTTTTTTTTATAATTTTAGTGTTTGAAAGGTTCGGGGCGTTTCGGGGTTTGTTAAGAGACGGTGTTTAGAGAAAGAGTATAACGCGAAGTTATAATCGTAGGAAATTCGTAGTAGTTTTTTTTCGTCGTTGGTTTCGTTTA GCGGGGAGAAAGGAGGGTCGTTTAGTTTTGCGTTTTGGGGCGTATCGAAGCGTCGGGATTTAAGAGGAGTAGGTAGGGACGGGGGTAGGGGAGGTCGCGGGGTAGTCGAGGAGCGTTTATATACGATTTTCGGAGTTCGAGGTTTTTTCGTGCGTTCGATTATAGATTAGAGGAGTTTTAGAAGAGGGCGAGATTAAAGCGTTCGTTTGGATGTTTTGTGCGTTTTTTATTTTGGTTTTTCGAAGATGTTTATTA ATTTGGCGATTAGTTGTTTTATGGTATTTTTCGGGTCGGATAGGTGTAGGAGGGTTTAGCGGTTTTAGGAGCGGAGTCGGTTTTGGTATTCGGAGTTTCGTTTTTTCG (SEQ ID NO. 67).

The synthetic sequence of the negative control region 11-14 is shown in SEQ ID NO. 68 (5’-3’):

TGTTTTTTTGTAGGTTGGGGATTTTTGTTATTGTATTGTGTTTGTTTTTTATTTAGTTTTGATATTTGTTTTATTTTGGGATGGTGGAAAAGATAGTTGAGTTTTTATTTTTTTTTATAATTTTAGTGTTTGAAAGGTTTGGGGTGTTTTGGGGTTTGTTAAGAGATGGTGTTTAGAGAAAGAGTATAATGTGAAGTTATAATTGTAGGAAATTTGTAGTAGTTTTTTTTTTTGTTGTTGGTTTTGTTTA GTGGGGAGAAAGGAGGGTTGTTTAGTTTTGTGTTTTGGGGTGTATTGAAGTGTTGGGGATTTAAGAGGAGTAGGTAGGGATGGGGGTAGGGGAGGTTGTGGGGTAGTTGAGGAGTGTTTATATATGATTTTTGGAGTTTGAGGTTTTTTTGTGTGTTTGATTATAGATTAGAGGAGTTTTAGAAGAGGGTGAGATTAAAGTGTTTGTTTGGATGTTTTGTGTGTTTTTTTTGGTTTTTTGAAGATGTTTATTA ATTTGGTGATTAGTTGTTTTATGGTATTTTTTGGGTTGGATAGGTGTAGGAGGGTTTAGTGGTTTTAGGAGTGGAGTTGGTTTTGGTATTTGGAGTTTTGTTTTTTTG (SEQ ID NO. 68).

The synthetic sequence of the positive control in regions 15 and 16 is shown in SEQ ID NO. 69 (5’-3’):

CGTATGCGTATAGACGATTCGAGTTTGTTTCGCGGTTGTTTAATTCGTTGAGAGTTGCGAGAAATCGAGTGAGAGAAAGTTTTGTAGTTTTTTCGATTTTATGTTTTTTTGGTATTAGGTATTCGTCGGGTCGTGGGGGGTTCGTAGTCGAACGTCGATTTTCGTTCGTATTGGGTTGGGAGTTTAGAGTCGCGCGTAGAATTCGGGTTGGTCGTAACGTTTGTGTTTTTAGCGGTGGTCGGGAATTTG GGATTAGGGTTATTTGAGTTGACGGGGTGGGGGCGGGTCGAGTGGGGTTGGAAGTTTGGAATTTAGTGGTAAGTAGGAGGGCGTAGGAGGTGGTAGTTAGGTAAGAGGTATTTTTATTTATTTAACGTTGGTTTGGGTCGTAATTTTATTTGGGAGTTTTTTTTTTCGGTGAGATAGAGATTCGGTAGAAGAAGCGGGAGGGGTTGGAGGTTGGTTTTTAGGTAGGTATTGTTCGGCGATTGGAGCGCGGATT TGGTTATTTGGGTGGGGTTGAGTGGGGGCGCGATTGTGAGTAGTAGTCGCGGGACGTTGCGAAGGGGCGGCGGTAATAGAGTACGGGCGGGGGTAGAAAAGAGGCGGCGGAGGGCGCGGTGGGGGAGCGCGAGGCGAGTGTTGAGAGAGTAGAAAGGATTTAAGTTTGAGGGGAGTAGAGAGGAAGAAGGGGTAACGCGAGAAATCGAATAGGAGTCGGCGTTTTTTGGTAAGGGAGGGCGGAGGCGCGC GGGAGAGAGGGAGAGGGAGGGCGGGGGGCGGGGGTAGGCGCGGGGAGAGGGGAGTATAATTCGTCGGTCGCGAGGAGCGGGGGTAGTTTCGGGTGTCGAGGTTTGTAGTTAGCGGTAAGCGGAGTTAGGTATTCGTTTAGATTGATAGTAGAGGCGGCGAAGGAGCGCGTAGTCGAGATTAGGCGTATAGAGTTCGGAGGCGGCGGCGGG (SEQ ID NO. 69).

The synthetic sequence of the negative control in regions 15 and 16 is shown in SEQ ID NO.70 (5’-3’):

TGTATGTGTATAGATGATTTGAGTTTGTTTTGTGGTTGTTTAATTTGTTGAGAGTTGTGAGAAATTGAGTGAGAGAAAGTTTTGTAGTTTTTTTGATTTTATGTTTTTTTGGTATTAGGTATTTGTTGGGTTGTGGGGGGTTTGTAGTTGAATGTTGATTTTTGTTTGTTATTGGGTTGGGAGTTTAGAGTTGTGTGTAGAATTTGGGTTGGTTGTAATGTTTGTGTTTTTAGTGGTGGTTGGGAATTTG GGATTAGGGTTATTTGAGTTGATGGGGTGGGGGTGGGTTGAGTGGGGTTGGAAGTTTGGAATTTAGTGGTAAGTAGGAGGTGTAGGAGGTGGTAGTTAGGTAAGAGGTATTTTTATTTATTTAATGTTGGTTTGGGTTGTAATTTTATTTGGGAGTTTTTTTTTGGTGAGATAGAGATTTGGTAGAAGAAGTGGGAGGGGTTGGAGGTTGGTTTTTAGGTAGGTATTGTTTGGTGATTGGAGTGTGGATT TGGTTATTTGGGTGGGGTTGAGTGGGGGTGTGATTGTGAGTAGTAGTTGTGGGATGTTGTGAAGGGGTGGTGGTAATAGAGTATGGGTGGGGGTAGAAAAGAGGTGGTGGAGGGTGTGGTGGGGGAGTGTGAGGTGAGTGTTGAGAGAGTAGAAAGGATTTAAGTTTGAGGGGAGTAGAGAGGAAGAAGGGGTAATGTGAGAAATTGAATAGGAGTTGGTGTTTTTTGGTAAGGGAGGGTGGAGGTGTGT GGGAGAGAGGGAGAGGGAGGGTGGGGGGTGTGGGGGTAGGTGTGGGGAGAGGGGAGTATAATTTGTTGGTTGTGAGGAGTGGGGGTAGTTTTGGGTGTTGAGGTTTGTAGTTAGTGGTAAGTGGAGTTAGGTATTTGTTTAGATTGATAGTAGAGGTGGTGAAGGAGTGTGTAGTTGAGATTAGGTGTATAGAGTTTGGAGGTGGTGGTGGG (SEQ ID NO. 70).

The synthetic sequence of the positive control in regions 17 and 18 is shown in SEQ ID NO.71 (5’-3’):

TTGAGCGGAGAGTGGGAGGGGAGGAAATCGAGGTGGATTGGGAATCCGGTTTGGTCGGTTTGTCGTTTGTTATTTGAGAGGCGAAGGGGTTGTAGCGGCCCGGTTTTTATTTAGGTTTTCGGTTTTGTTGAGGGGGTGGGGGGTTTCGTTTGTTAGTGGGACGCGGATATGGATTAGGTTTTTTTTATTTTTTAGATCGAGAAGGCGTAGTTGAGTCGTTCGTGAGTTATATAGTTGTAG TTTGTTATTTTGGAGTTTAGTTCGTCGTTTTGGAGGATTTGGTAGAGGAAGTCGATGTATTTGGTCGTTAGTTTGAGGGTTTGAATTTTGTTTAGTTTGTTCGAGGGTAGCGTGGGGATGATTTTTCGTAGCGCGGCGAACGTTCGTTTAGCGATTGGGTGCGTTGGCGTTTTCGTACGTTGGTTATGATTCGTTGCGTTTGTAGTTTTTCGTAAGATTGCGGATTTTCGTCGTCGTTGTTGTTGTCG TCGTCGTCGTTCGCGTCGTCGTCGTCGTTATAGTTCGTAGATTTTTTGTC (SEQ ID NO. 71);

The synthetic sequence of the negative control in regions 17 and 18 is shown in SEQ ID NO.72 (5’-3’):

TTGAGTGGAGAGTGGGAGGGGAGGAAATTGAGGTGGATTGGGAATTGTGGTTTGGTTGGTTTGTTGTTTGTTATTTGAGAGGTGAAGGGGTGTAGTGGTGTGGTTTTTATTTAGGTTTTTGGTTTTGTTGAGGGGGTGGGGGGTTGTTTGTTAGTGGGATGTGGATATGGATTAGGTTTTTTTTATTTTTTAGATTGAGAAGGTGTAGTTGAGTTGTTTGTGAGTTATATAGTTGTAGTTTTG TTATTTTGGAGTTTAGTTTTGTTGTTTTGGAGGATTTGGTAGAGGAAGTTGATGTTATTTGGGTTGTTAGTTTGAGGGTTTGAATTTTGTTTAGTTTGTTTGAGGGTAGTGTGGGGATGATTTTTTGTAGTGTGGTGAATGTTTTGTTTAGTGATTGGGTGTGTTGGTTTTTGTATGTTGGTTATGATTTGTTGTGTTTGTAGTTTTTTGTAAGATTGTGGATTTTTGTTGTTGTTGTTGTTGTTGTTG TTGTTGTTTGTGTTGTTGTTGTTGTTATAGTTTGTAGATTTTTTGTC (SEQ ID NO. 72).

The synthetic sequence of the positive control in region 19 is shown in SEQ ID NO.73 (5’-3’):

TTGTTCGCGGGTTTTCGTTTGATCGTAGTTTCGAGATCGTCGCGTATTTTTTTTTACGTTTTTTTGGCGTGGTTATCGGATTTTTTTGGTTTAGTTTTAGGCGGATTTTTTTTTTATCGCGCGATTTCGTTTTTTTTAGTATTTTAGGGTGAGTTTAGTTTAGATTATTATTCGGAAAGTTTTTAAAAGTTTTAGTTTAGCGTTGAAGTAACGGGATTATGTTTAGTTTTAGGTTTCGGAGTAG( SEQ ID NO. 73).

The synthetic sequence of the negative control in region 19 is shown in SEQ ID NO.74 (5’-3’):

TTGTTTGTGGGTTTTTGTTTGATTGTAGTTTTGAGATTGTTGTGTATTTTTTTTTATGTTTTTTTGGTGTGGTGTTATTGGATTTTTTTGGTTTAGTTTTAGGTGGATTTTTTTTTTATTGTGTGATTTTGTTTTTTTTAGTATTTTAGGGTGAGTTTAGTTTAGATTATTATTTGAAAGTTTTTAAAAGTTTTAGTTTAGTGTTGAAGTAATGGGATTATGTTTAGTTTTAGGTTTTGGAGTAG (SEQ ID NO.74).

The synthetic sequence of the positive control in region 20 is shown in SEQ ID NO.75 (5’-3’):

CGAACGTTTTAGTGTTTTCGATTTTGGGTAGCGGGGATTCGAGTAGGCGTTTTTTATTGATGGTTTTAGAACGTGGGTGGGGGAAGGTGTGTGAGGACGGGAAGACGTCGTATTTATTTGAGTTGGCGTTTTTAGAGTGGTCGTTGTTATTAGATTTTGCGGGTAGAGTTGGGTCGGGAGCGACGGGCGATATTGGTAGGGATTCGGGGATAGCGGTTTTTTTTAGGTTTGACGTGGGTTTTTTTT AGGGCGGCGTCGTTAAGGTTTAGACGTTTTCGTGTAGGAGGGACGACGATTTTTTTTACGTTTTCGTGGTTTTTAATTCGGCG (SEQ ID NO. 75).

The synthetic sequence of region 20 negative control is shown in SEQ ID NO.76 (5’-3’):

TGAATGTTTTAGTGTTTTTGATTTTGGGTAGTGGGGATTTGAGTAGGTGTTTTTTATTGATGGTTTTAGAATGTGGGTGGGGGAAGGTGTGTGAGGAIGGGAAGATGTTGIATTTATTTGAGTTGGTGTTTTTAGAGTGGTTGTTGTTATTAGATTTTGTGGGTAGAGTTGGGTTGGGAGTGATGGGTGATATTGGTAGGGATTTGGGGATAGTGGTTTTTTATTTTAGGTTTGATGTGGGTTTTTT AGGGTGGTGTTGTTAAGGTTTAGATGTTTTTGTGTAGGAGGGATGATGATTTTTTTTATGTTTTTGTGGTTTAATTTGGTG (SEQ ID NO. 76).

The artificial sequences corresponding to SEQ ID NO. 61-76 were cloned into the vector pUC57 to form artificial plasmids. Each plasmid was diluted to 10 ³ copies/microliter to form a positive control.

3. PCR reaction

Using β-actin as the internal reference gene, the PCR reaction system is shown in Table 2. β-actin is used as the internal reference gene, where the β-actin upstream primer is: AAGGTGGTTGGGTGGTTGTTTTG (SEQ ID NO.77); the β-actin downstream primer is: AATAACACCCCCACCCTGC (SEQ ID NO.78); the β-actin probe is: GGAGTGGTTTTTGGGTTTG (SEQ ID NO.78) IDNO.79).

The reporter group at the 5' end of the probe that detects the target region is FAM, and the quencher group at the 3' end is MGB. The reporter group at the 5' end of the β-actin probe is VIC, and the quencher group at the 3' end is BHQ1.

Table 2

Components Specification Volume(μL) Buffer (containing magnesium ions) 5× 10 dNTPs 2.5mM each 5 Detection region upstream primers 10μM 1 Detection region downstream primers 10μM 1 detection area probe 10μM 1 β-actin upstream primer 10μM 1 β-actin downstream primer 10μM 1 β-nctin probe 10μM 1 hot start DNA polymerase 5U/μL 0.5 Sample DNA to be tested / 10 purified water / Replenish to 50

As shown in Table 2, the methylation status of regions 1-20 of each sample was detected separately, that is, in one PCR tube, only the primer probe of one region was added, and the primer probe of β-actin was also added. Three duplicate holes were set for each test.

PCR reaction conditions are shown in Table 3 below.

table 3

Ct value reading: After the PCR is completed, adjust the baseline individually for each gene. Set the fluorescence value 1-2 cycles before the minimum Ct value of the sample in a PCR as the baseline value, and set the threshold at the S-shaped amplification curve. At the inflection point, the PCR instrument software will automatically obtain the Ct value of each gene in the sample.

Quality control: During each test, the negative control and the positive control are tested simultaneously. The concentration is 10 ³ copies/microliter. The negative control must have no amplification, the positive control must have an obvious exponential growth period, and the positive control must have three replicates. The average hole Ct value should be between 26-30. The Ct value of the internal reference gene in the sample should be ≤33. When the negative control, positive control and internal reference gene all meet the above requirements, it means that the experiment is valid and the next step of determining the sample results can be carried out. Otherwise, the current experiment will be invalid and must be retested.

Results analysis and interpretation method: Calculate the difference between the Ct value of each sample detection target region and the internal reference gene, that is, the ΔCt value, use SPSS 22.0 software to perform ROC analysis, set the state variable of the urothelial cancer sample as "1", and set the normal The state variable of the sample is positioned at "0". When only analyzing the diagnostic performance of a single detection area, click "Analysis" - "ROC Curve", specify the values of the test variable and the state variable, set the value of the state variable to 1, and Select "Smaller test results represent a more definite test" to obtain the ROC analysis results, select ΔCt when the Youden index is the largest as the cutoff value, and obtain the average AUC value, sensitivity and specificity values, and calculate the sensitivity and specificity. The method is:

Samples with ΔCt less than or equal to the cutoff value are methylation positive samples, samples with ΔCt greater than the cutoff value are methylation negative samples, sensitivity is the proportion of methylation positive samples in cancer samples, and specificity is methylation in healthy samples. Negative proportion.

When analyzing the joint diagnostic performance of multiple detection areas, first perform a binary or multivariate logistic analysis on the ΔCt values of multiple areas to obtain a probability value, and then use the probability value as the test variable to perform ROC analysis. The software setting process is the same as above. Analysis process of a single detection area.

Experimental example 2

A total of 66 cases of urothelial cancer (including 1 case of renal pelvis cancer, 1 case of ureteral cancer and 64 cases of bladder cancer) and 89 healthy human urine samples were collected from a hospital in Beijing. These samples were used as training samples. According to the embodiment, The method described in 1 performs genome extraction, bisulfite conversion, and uses the converted DNA as a template to detect the methylation status of region 1 to region 20 respectively. Use SPSS software to perform ROC analysis on the ΔCt values of urothelial cancer samples and healthy human samples, and draw a ROC curve, as shown in Figure 1. Record the sensitivity, specificity and average AUC value of individual areas in areas 1-20, see Table 4.

Table 4

It can be seen from the results in Table 4 that the four regions of the NBR1 gene (regions 1-4) have a sensitivity of more than 50% and a specificity of more than 97% for the detection of urothelial cancer; the two regions of ZNF551 (regions 5 and 4) have a sensitivity of more than 50% and a specificity of more than 97%. The detection sensitivity of area 6) for urothelial cancer is above 45%, and the specificity is above 98%; the detection sensitivity of 4 areas of CYTH2 (areas 7-10) for urothelial cancer is above 55%. The specificities are all above 96%; the detection sensitivity of the four regions of ARL5C (regions 11-14) for urothelial cancer is above 60%, and the specificity is above 95%. The average AUC values of the four genes NBR1, ZNF551, CYTH2 and ARL5C are significantly higher than those of the POU4F2, TWIST1, VIM and ZNF154 genes. Among the latter four genes, except for region 18 on the TWIST1 gene, the AUC value of the remaining regions is greater than 0.7. The AUC values are all below 0.7, the detection sensitivity of areas 15 to 19 for urothelial cancer is less than 50%, and the detection specificity of area 20 is less than 90%. That is to say, the methylation level of the marker according to the present invention can distinguish healthy samples from urothelial cancer samples, and its detection sensitivity and specificity are both higher than existing gene markers for urothelial cancer.

In general, the average AUC values of regions 1-14 are above 0.7, among which the average AUC values of regions 12 and 1 are above 0.8. Regions 1-14 can be detected in 1 renal pelvis cancer sample and 1 ureteral cancer sample. The above results indicate that regions 1-14 are hypermethylated in urothelial cancer samples and hypomethylated in healthy human samples. It uses the methylation level of regions 1-14 as the detection target and can distinguish urothelial cancer samples from healthy human samples with high sensitivity and specificity.

Experimental example 3

A total of 66 cases of urothelial cancer (including 1 case of renal pelvis cancer, 1 case of ureteral cancer and 64 cases of urothelial cancer) and 89 healthy human urine samples (same as Experimental Example 1) were collected from a hospital in Beijing. The method described in 1 performs genome extraction, bisulfite conversion, and uses the converted DNA as a template to detect the methylation status of region 1 to region 14 respectively. Use SPSS software to analyze the diagnostic performance of two or more regions and draw a ROC curve, as shown in Figure 1. Record the sensitivity, specificity and average AUC value of the regional combination, see Table 5.

table 5

It can be seen from the results in Table 5 that when two or more genes are used for joint detection, the sensitivity and average AUC value are significantly improved compared with single-gene detection, and the specificity remains above 90%. The detection sensitivities of area 1+area 6, area 1+area 8, area 6+area 8, area 6+area 11, and area 8+area 11 are all above 70%, and the detection sensitivity of area 1+area 11 is also close to 70%. , the detection sensitivity of the four genes was 86.4%.

Example 4

A total of 32 cases of bladder cancer and 50 cases of healthy human urine samples were collected from a hospital in Beijing. These samples were used as test samples. Genome extraction and bisulfite conversion were performed according to the method described in Example 1, and the converted DNA was used as a template to detect the methylation status of Region 1 to Region 14 respectively, and the ΔCt value of each region in each sample was obtained. The yin and yang of each region in the sample was determined based on the cutoff value of each region obtained in Example 2. sex, that is, when ΔCt is greater than the cutoff value, it is judged as methylation negative, and when ΔCt is less than or equal to the cutoff value, it is judged as methylation positive. The sensitivity and specificity of regions 1-14 are calculated based on the proportion of methylation positive and negative respectively. The results are shown in Table 6.

Table 6

According to the analysis results of the test samples, the detection sensitivity of 32 cases of bladder cancer in areas 1-14 is 53.13%-71.88%, and the specificity is 94%-100%. The test sample detection results are consistent with the conclusions obtained from the training samples.

The technical features of the above-described embodiments can be combined in any way. To simplify the description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, All should be considered to be within the scope of this manual.

The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined by the appended claims.

Claims (10)

1. Use of a reagent for detecting the methylation level of a target gene comprising the CYTH2 gene and/or the ZNF551 gene in the manufacture of a kit for detecting urothelial cancer.

2. The use according to claim 1, wherein the target gene further comprises an ARL5C gene.

3. The use according to claim 1 or 2, wherein the reagent comprises at least one of a primer pair and a probe for detecting the methylation level of the target gene.

4. The use according to claim 3, characterized in that the primer pair comprises a primer pair for amplifying a CYTH2 gene CpG island and/or for amplifying a ZNF551 gene CpG island;

the primer pair for amplifying the CYTH2 gene CpG island is selected from at least one group of SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.22 and SEQ ID NO.23, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.28 and SEQ ID NO. 29;

the primer pair for amplifying the ZNF551 gene CpG island is selected from at least one group of SEQ ID NO.13 and SEQ ID NO.14, SEQ ID NO.16 and SEQ ID NO. 17.

5. The use according to claim 4, wherein the probe comprises a probe for detecting a CpG island of the CYTH2 gene and/or a CpG island of the ZNF551 gene,

wherein, the probe for detecting the CYTH2 gene CpG island is at least one selected from SEQ ID NO.21, SEQ ID NO.24, SEQ ID NO.27 and SEQ ID NO. 30;

the probe for detecting the ZNF551 gene CpG island is selected from at least one of SEQ ID NO.15 and SEQ ID NO. 18.

6. The use according to any one of claims 4 to 5, wherein the primer pair further comprises a primer pair for amplifying an ARL5C gene CpG island;

wherein, the primer pair for amplifying the ARL5C gene CpG island is selected from at least one group of SEQ ID NO.31 and SEQ ID NO.32, SEQ ID NO.34 and SEQ ID NO.35, SEQ ID NO.37 and SEQ ID NO.38, SEQ ID NO.40 and SEQ ID NO. 41;

preferably, the probe further comprises a probe for detecting the CpG island of the ARL5C gene; the probe for detecting the ARL5C gene CpG island is at least one selected from the group consisting of SEQ ID NO.33, SEQ ID NO.36, SEQ ID NO.39 and SEQ ID NO. 42.

7. The use of claim 1, wherein the kit further comprises at least one of a nucleic acid extraction reagent, a nucleic acid conversion reagent, a PCR reaction reagent, a positive control sample, and a negative control sample.

8. The use according to claim 1, wherein the urothelial cancer comprises at least one of renal pelvis cancer, ureter cancer and bladder cancer.

9. A kit for the detection of urothelial cancer prepared according to the use of any one of claims 1-8.

10. A computer readable storage medium for storing computer instructions, programs, code sets or instruction sets which when executed on a computer cause the computer to perform all the steps of predicting urothelial cancer, the all the steps comprising:

methylation information of CpG islands of ZNF551 and/or CYTH2 genes in a sample to be detected is obtained, wherein the sample to be detected is a DNA sample after nucleic acid conversion treatment;

and judging the type of the sample to be detected as a urothelial cancer sample or a health sample according to the methylation information.