Methylation marker combination for diagnosis or prediction of colorectal cancer and application, kit and computer readable storage medium

Abstract

The application discloses a methylation molecular marker combination for colorectal cancer diagnosis or prediction, which is composed of the following two methylation molecular markers respectively located at human SLIT2 and PAX5 genes: chr4:20253208-20253408 and chr9:37002603-37002803, and is specifically positioned by means of hg38. The application also discloses application of the methylation molecular marker combination, a related kit and an analysis processing program recorded by a computer readable storage medium. The methylation levels of the SLIT2 and PAX5 genes serve as markers for early diagnosis or prediction of the colorectal cancer, and enrich the selection of the skilled in the art.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology and gene detection, and particularly relates to a methylation marker combination for colorectal cancer diagnosis or prediction, an application, a kit and a computer readable storage medium. BACKGROUND

[0002] Colorectal cancer (CRC) is a common malignant tumor, which refers to a malignant tumor occurring in the lower digestive tract of the human body, and is one of the main cancers threatening human health.

[0003] According to the multi-stage theory of the carcinogenic process, the occurrence of CRC shows a phased evolution from normal mucosa hyperplasia, polyp adenoma formation, adenoma carcinogenesis to invasive metastasis. It takes 10-15 years for polyp adenoma to evolve into CRC, and the cure rate of early CRC is more than 90%, while the cure rate of late CRC is less than 10%. Therefore, it is necessary to identify the risk of CRC precancerous lesions, screen and diagnose early to reduce the mortality rate of CRC.

[0004] The CRC and early diagnosis and treatment program published in 2024 mentions that CRC precancerous lesions include adenomas with a diameter of ≥10 mm, adenomas with a villous structure of ≥25% (i.e., villous adenomas or mixed adenomas), and other lesions with high-grade intraepithelial neoplasia. It is recommended that all adenomas, polyps, especially CRC precancerous lesions and CRC patients, receive standardized treatment as soon as possible.

[0005] Colonoscopy is the gold standard for colorectal cancer screening, but its invasiveness and complex bowel preparation result in low compliance of colonoscopy screening in Chinese people. In addition, the demand for colonoscopy is large, and with the aging of the population, the number of elderly people and people over 40 years old is increasing, and the number of colonoscopy examinations is showing an explosive growth. Large-scale application of colonoscopy for screening will also cause great waste of resources. The traditional screening program uses a two-step screening mode of questionnaire survey combined with two fecal occult blood tests (FIT). If any one item is positive, it is determined as a primary screening positive, which indicates a high-risk group and needs to receive colonoscopy examination. This screening has the problems of high false positive rate and low colorectal cancer detection rate, and the screening mode leads to insufficient manpower investment in hospitals, resulting in slow progress of the project.

[0006] Peripheral blood is one of the most studied biological sample types, and blood testing can be a popular choice for people who are asymptomatic, at moderate risk, unwilling to undergo a stool test or endoscopy. The current products on the market based on blood DNA testing for the auxiliary diagnosis of colorectal cancer have a generally low sensitivity of less than 85%, which cannot meet the clinical needs. Clinical practice shows that blood Septin9 methylation detection is mainly limited to its relatively low sensitivity in identifying colorectal cancer and precancerous lesions (adenomas), and the sensitivity for advanced adenomas is only 7.9% to 38.7%.

[0007] Colorectal cancer screening technology based on fecal DNA detection mainly targets the genetic mutations and / or methylation of exfoliated cells in the colorectum, overcomes the main defect of detecting trace amounts of bleeding, has single-target and multi-target schemes, can also be combined with FIT detection, and has the advantages of not requiring special equipment, not requiring dietary restrictions, and being non-invasive. Although there has been a significant improvement in the sensitivity and specificity of colorectal cancer, the detection rate for 0-II early-stage cancer is still below 90%, and the detection rate for advanced adenomas such as precancerous lesions and high-grade intraepithelial neoplasia is even lower, all below 65% or even lower. Although fecal DNA detection can be performed at home, fecal sampling is relatively private and inconvenient, which is different from the general medical habits of the public. The public needs very long health education before accepting fecal DNA detection, and there are still some problems in detection compliance during screening promotion.

[0008] Therefore, it is an urgent problem to develop blood samples based on the highest user compliance in clinical practice and to explore new markers for colorectal cancer with high sensitivity.

[0009] Methylation of cytosine on DNA is a covalent DNA "acquired" modification. DNA methylation is carried out by DNA methyltransferases (DNMTs). DNMTs can transfer a methyl group from one S-adenosyl methionine to the C-5 position of cytosine. DNA methylation almost specifically occurs at CpG doublets, and CpG doublets are unevenly distributed in the human genome, with the region generally referred to as a CpG island. Such CpGs exist in repeat sequences and the regulatory regions of many genes 5' in the human genome. Abnormal DNA methylation in tumors includes hypomethylation (or demethylation) and hypermethylation, and hypermethylation includes but is not limited to tumor suppressor genes, and hypomethylation includes but is not limited to proto-oncogenes.

[0010] The development of tumors in all aspects is likely to be associated with methylation changes in DNA, involving cell cycle regulation, DNA damage repair, biochemical metabolism of carcinogenic compounds, apoptosis and angiogenesis. Different types of tumors may have a specific group of hypermethylated tumor suppressor genes, hypomethylated oncogenes, i.e. a specific methylation profile of a cancer type, and the type of cancer can be identified according to the methylation profile.

[0011] Methylation enrichment techniques are an analysis method for studying methylation modifications on DNA. DNA methylation is an important epigenetic modification involving the addition of a methyl group to the cytosine ring in DNA molecules. This modification plays a key role in regulating gene expression, cell differentiation, genome stability and other biological processes. Therefore, understanding the status of DNA methylation is very important for understanding biological processes and the development of diseases.

[0012] Common methylation enrichment techniques include methylation-specific PCR (MSP), methylation-sensitive restriction enzyme digestion, methylated DNA immunoprecipitation (MeDIP), methylated DNA immunoprecipitation sequencing (MeDIP-Seq) and MBD-Seq (Methyl-CpG Binding Domain sequencing), etc., wherein:

[0013] MSP uses methylation-specific primers to selectively amplify methylated DNA fragments by PCR. It is simple, fast, and suitable for specific CpG site analysis, but cannot provide whole-genome methylation information and is only suitable for previously determined target regions.

[0014] Methylation-sensitive restriction enzyme digestion uses the difference in sensitivity of restriction enzymes to DNA sequences to distinguish between methylated and non-methylated DNA regions. This method is based on the principle that DNA methylation affects the sensitivity of base pairs on the cytosine ring to restriction enzymes. This technique does not require the use of expensive sequencing technology and can be analyzed by gel electrophoresis and other methods. However, it cannot provide high-resolution information for individual CpG sites and usually provides overall regional methylation status. At the same time, it is limited by the specificity of the selected restriction enzymes, which may miss or overestimate some methylation sites. In addition, it cannot directly distinguish between 5-methylcytosine and other forms of DNA modification.

[0015] MeDIP-Seq uses methylation DNA antibody to selectively enrich methylated DNA fragments, and then analyzes the enrichment by high-throughput sequencing technology. The entire methylated genomic region can be enriched, which is suitable for whole genome methylation analysis. However, it cannot provide high-resolution information of individual CpG sites.

[0016] MBD-Seq uses methylation DNA binding protein (such as MBD2 or MBD3) to enrich methylated DNA fragments, and then analyzes by sequencing. It can provide higher enrichment efficiency and is suitable for whole genome methylation analysis. However, similar to MeDIP-Seq, it cannot provide high-resolution information of individual CpG sites.

[0017] Methylation treatment is also known as methylation conversion. Common methylation treatment sequencing techniques include bisulfite sequencing (BS-seq). BS-seq uses bisulfite to treat DNA, converting non-methylated cytosine to uracil, while methylated cytosine is not affected, and then analyzes by sequencing. It can provide high-resolution information of individual CpG sites and can perform whole genome methylation analysis. However, the experimental steps are complicated.

[0018] The current mainstream methylation analysis method is bisulfite treatment, which includes denaturation, deamination, and desulfonation. DNA is first denatured into single strands, then subjected to high temperature, high salt, acidic, and alkaline environments, and the resulting converted DNA has the following characteristics: single-stranded, double-stranded mixed, fragment notching, gap damage, and uracil state nucleotides. This process generally results in the loss of 90% of the DNA template, and a large amount of methylation information cannot be detected by subsequent processes. At the same time, during base conversion treatment, there are cases of incomplete or excessive sequence conversion, resulting in artificial bias, which is further amplified by subsequent PCR amplification, causing inaccurate signals. Therefore, the methylation markers obtained based on bisulfite treatment currently have the problem of low sensitivity, especially in blood samples, where the number of free DNA fragments is already limited, and after bisulfite treatment, the difficulty of detecting methylation levels is greatly increased.

[0019] Although the sensitivity to colorectal cancer can be improved by increasing the number of methylation genes based on bisulfite treatment, such as the intestinal cancer 3 gene methylation joint detection of Beijing Aikun Medical Technology Co., Ltd., which was approved by the National Medical Products Administration (NMPA) in 2022. The clinical trial sensitivity of blood sample cfDNA treated by bisulfite is 84.75% (328 / 387), but the sensitivity still cannot meet the actual needs of clinical practice. Therefore, it is the most urgent need to find colorectal cancer related gene methylation markers in human blood samples based on non-bisulfite treatment and effectively detect the methylation level changes. SUMMARY

[0020] One of the technical problems to be solved by the present application is to provide a methylation molecular marker combination for diagnosing or predicting colorectal cancer, which consists of the following two methylation molecular markers located in human SLIT2 (Slit Guidance Ligand 2) and PAX5 (Paired Box 5) genes, respectively: chr4:20253208-20253408 and chr9:37002603-37002803, which are specifically located by hg38.

[0021] The second technical problem to be solved by the present application is to provide a use of a reagent for detecting the methylation molecular marker combination as described above in the preparation of a product for diagnosing or predicting colorectal cancer.

[0022] In some embodiments, the reagent for detecting the methylation molecular marker combination as described above comprises a reagent used in any one or more of the following methods, including at least one of methylation DNA immunoprecipitation fluorescence quantitative PCR method, bisulfite conversion fluorescence quantitative PCR method, bisulfite conversion sequencing method, methylation chip sequencing, and methylation specific PCR method.

[0023] In some embodiments, the reagent for detecting the methylation molecular marker combination as described above comprises primer and probe combination a and / or primer and probe combination b;

[0024] Primer and probe combination a: a primer pair as shown in SEQ ID No. 1 and SEQ ID No. 2 and a Taqman MGB probe as shown in SEQ ID No. 3 for fluorescence quantitative PCR amplification of chr4:20253208-20253408; a primer pair as shown in SEQ ID No. 4 and SEQ ID No. 5 and a Taqman MGB probe as shown in SEQ ID No. 6 for fluorescence quantitative PCR amplification of chr9:37002603-37002803;

[0025] Primer and probe combination b: primer pair as shown in SEQ ID No. 7 and SEQ ID No. 8 and Taqman MGB probe as shown in SEQ ID No. 9 for the fluorescent quantitative PCR amplification of chr4:20253208-20253408; primer pair as shown in SEQ ID No. 10 and SEQ ID No. 11 and Taqman MGB probe as shown in SEQ ID No. 12 for the fluorescent quantitative PCR amplification of chr9:37002603-37002803.

[0026] A third technical problem to be solved by the present application is to provide a kit for diagnosing or predicting colorectal cancer. The kit is used for detecting a methylation molecular marker combination; the methylation molecular marker combination consists of the following 2 methylation molecular markers respectively located at human SLIT2 and PAX5 genes: chr4:20253208-20253408 and chr9:37002603-37002803, which are specifically located by hg38;

[0027] The kit comprises primer and probe combination a and / or primer and probe combination b;

[0028] Primer and probe combination a: primer pair as shown in SEQ ID No. 1 and SEQ ID No. 2 and Taqman MGB probe as shown in SEQ ID No. 3 for the fluorescent quantitative PCR amplification of chr4:20253208-20253408; primer pair as shown in SEQ ID No. 4 and SEQ ID No. 5 and Taqman MGB probe as shown in SEQ ID No. 6 for the fluorescent quantitative PCR amplification of chr9:37002603-37002803;

[0029] Primer and probe combination b: primer pair as shown in SEQ ID No. 7 and SEQ ID No. 8 and Taqman MGB probe as shown in SEQ ID No. 9 for the fluorescent quantitative PCR amplification of chr4:20253208-20253408; primer pair as shown in SEQ ID No. 10 and SEQ ID No. 11 and Taqman MGB probe as shown in SEQ ID No. 12 for the fluorescent quantitative PCR amplification of chr9:37002603-37002803.

[0030] In some embodiments, the enrichment of methylated fragments is performed by using the Methylated DNA Immunoprecipitation (MeDIP) technology, and then using the enriched DNA as a template for performing the quantitative PCR under the condition of primer and probe combination a; the kit further comprises a methylated DNA antibody selected from one of 5-methylcytosine antibody, 5-methylcytosine (5-mC) antibody, 5-hydroxymethylcytosine (5-hmC) antibody, 5-formylcytosine (5-fC) antibody, 5-carboxylcytosine (5-caC) antibody.

[0031] In some embodiments, one or more of the following are further included: Rapid Taq Master Mix, reagents required for methylation enrichment based on the principle of 5-methylcytosine antibody, and reagents required for methylation conversion based on the principle of bisulfite conversion.

[0032] In some embodiments, the 3' end of the Taqman MGB probe carries a MGB and a fluorescence quenching group, and the 5' end carries a fluorescence group; the combination of the fluorescence quenching group and the fluorescence group is selected from BHQ1 or NFQ and FAM, BHQ2 and VIC or HEX, BHQ2 and Cy3, BHQ2 and Cy5.

[0033] In some embodiments, the two Taqman MGB probes in primer and probe combination a respectively carry different fluorescence quenching group and fluorescence group combinations, so that in the fluorescence quantitative PCR amplification reaction with two Taqman MGB probes at the same time, the corresponding Ct value can be read according to the difference in fluorescence color.

[0034] In some embodiments, the two Taqman MGB probes in primer and probe combination b respectively carry different fluorescence quenching group and fluorescence group combinations, so that in the fluorescence quantitative PCR amplification reaction with two Taqman MGB probes at the same time, the corresponding Ct value can be read according to the difference in fluorescence color.

[0035] In some embodiments, the two Taqman MGB probes of primer and probe combination a carry the same fluorescence quenching group and fluorescence group combination.

[0036] In some embodiments, the two Taqman MGB probes of primer and probe combination b carry the same fluorescence quenching group and fluorescence group combination.

[0037] The fourth technical problem solved by the present application is to provide a computer readable storage medium comprising a program capable of being executed by a processor to realize analysis and processing of fluorescence quantitative PCR detection data of the methylation molecular marker combination as described above, so as to obtain a colorectal cancer determination result, comprising the following steps:

[0038] Ct obtained by fluorescence quantitative PCR under the condition of primer and probe combination a, using formula I and formula II to process and judge the detection result SLIT2 , Ct PAX5 .

[0039] Formula I: logistic scores = e k / (1+e k )

[0040] Formula II: k = -2.478 x Ct SLIT2 -3.076 x Ct PAX5 + 203.157

[0041] logistic scores score ≤800 is determined as negative colorectal cancer, and logistic scores score >800 is determined as positive colorectal cancer.

[0042] The Ct value obtained by fluorescence quantitative PCR detection result is >45 or no Ct value is detected, and the Ct value is all taken as 45 so as to be substituted into formula II.

[0043] Primer and probe combination a: primer pair as shown in SEQ ID No. 1 and SEQ ID No. 2 and Taqman MGB probe as shown in SEQ ID No. 3, used for fluorescence quantitative PCR amplification of chr4:20253208-20253408 to obtain Ct SLIT2 ; primer pair as shown in SEQ ID No. 4 and SEQ ID No. 5 and Taqman MGB probe as shown in SEQ ID No. 6, used for fluorescence quantitative PCR amplification of chr9:37002603-37002803 to obtain Ct PAX5 .

[0044] The present application also provides another computer readable storage medium comprising a program capable of being executed by a processor to realize analysis and processing of fluorescence quantitative PCR detection data of the methylation molecular marker combination as described above, so as to obtain a colorectal cancer determination result, comprising the following steps:

[0045] Ct obtained by fluorescence quantitative PCR under the condition of primer and probe combination b with DNA converted by bisulfite as template SLIT2 Ct PAX5 , and the detection result is processed and judged by using formula I and formula III.

[0046] Formula I: logistic scores = e k / (1+e k )

[0047] Formula III: k = -0.36*Ct SLIT2 -1.373*Ct PAX5 +67.241

[0048] If the logistic scores score is less than or equal to 725, it is determined to be negative for colorectal cancer, and if the logistic scores score is greater than 725, it is determined to be positive for colorectal cancer.

[0049] If the Ct value obtained by fluorescence quantitative PCR detection is greater than 45 or no Ct value is detected, the Ct value is taken as 45 to be substituted into formula III.

[0050] Primer and probe combination b: the primer pair shown as SEQ ID No. 7 and SEQ ID No. 8 and the Taqman MGB probe shown as SEQ ID No. 9 are used for fluorescence quantitative PCR amplification of chr4:20253208-20253408 to obtain Ct SLIT2 ; the primer pair shown as SEQ ID No. 10 and SEQ ID No. 11 and the Taqman MGB probe shown as SEQ ID No. 12 are used for fluorescence quantitative PCR amplification of chr9:37002603-37002803 to obtain Ct PAX5 .

[0051] In some embodiments, the fluorescence quantitative PCR detection data of the program analysis processing of the computer readable storage medium is from the following fluorescence quantitative PCR reaction: the reaction system is 35 μL; the reaction reagent used is 2x Rapid TaqMaster Mix; and the reaction condition is 95℃ for 5 minutes, 95℃ for 15 seconds, 60℃ for 40 seconds, and amplification for 45 cycles.

[0052] Compared with the prior art, the present application has the following technical effects:

[0053] The application provides methylation molecular markers of SLIT2 and PAX5 gene methylation regions (chr4:20253208-20253408 and chr9:37002603-37002803, which are specifically located by hg38) for diagnosis (including early diagnosis and auxiliary diagnosis) or prediction of colorectal cancer, enriching the selection of those skilled in the art.

[0054] The application adopts methylation DNA immunoprecipitation technology combined with fluorescence quantitative PCR to detect the methylation level of the SLIT2 and PAX5 gene methylation regions, has high sensitivity and strong specificity, and has very important clinical application value.

[0055] The application can realize early detection of colorectal cancer based on the methylation level of specific regions of the SLIT2 and PAX5 genes, and provides more biomarker (the methylation molecular marker is one of the biomarkers) selection for screening and early diagnosis of colorectal cancer.

[0056] The concept, specific structure and generated technical effects of the application will be further described below in combination with the drawings, so as to fully understand the purposes, characteristics and effects of the application. BRIEF DESCRIPTION OF DRAWINGS

[0057] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0058] Figure 1 The SLIT2 and PAX5 methylation gene detection process in the embodiment 2 of the application is shown.

[0059] Figures 2A-2D An amplification curve graph of qPCR detection based on the methylation DNA immunoprecipitation enrichment method of a sample (as shown in Figure 2A and 2B ) and an amplification curve graph of qPCR detection based on the bisulfite conversion treatment (as shown in Figure 2C and 2D ) are shown.

[0060] Figure 3A The ROC curve of qPCR detection based on the methylation DNA immunoprecipitation enrichment method of 122 samples is shown.

[0061] Figure 3B The ROC curve of qPCR detection based on the bisulfite conversion treatment of 122 samples is shown. DETAILED DESCRIPTION

[0062] For the convenience of those skilled in the art, some terms appearing in the present text are explained and illustrated.

[0063] In the present text, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" can be read to include two or more agents.

[0064] In the present text, the terms "comprising", "including" or "containing" mean that the listed values, steps or components are included, but do not exclude other values, steps or components.

[0065] In the present text, "individual" or "patient" are used interchangeably and refer to a vertebrate, preferably a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples.

[0066] Numerical ranges in the present text are approximate, and thus include values outside the stated range, unless otherwise indicated. Numerical ranges include all values from and including the lower and to the upper range limits. In this context, a range includes each individual numerical value, e.g. a range of 1 to 10 includes each individual number from 1 to 10. This range is approximate because the described ranges include all values up to and including the upper and lower limits of the range. However, it specifically excludes any number or value outside that range.

[0067] The term "AUC" is an abbreviation of "Area Under the Curve". Specifically, it refers to the area under the Receiver Operating Characteristic (ROC) curve. The ROC curve is a plot of the true positive rate versus the false positive rate for the different possible cut points of a diagnostic test. It shows the balance between sensitivity and specificity for a chosen cut point (any increase in sensitivity will be accompanied by a decrease in specificity). The area under the ROC curve (AUC) is a measure of a diagnostic test (the larger the area, the better the test; the best is 1; a random test will have an ROC curve lying on the diagonal with an area of 0.5; reference: J. P. Egan. (1975) Signal Detection Theory and ROC Analysis, Academic Press, New York).

[0068] To detect the level change of colorectal cancer specific methylation genes in ctDNA, mine new colorectal cancer methylation markers with higher sensitivity and specificity, and construct an early screening and early diagnosis model, the inventors of the present application have made a lot of efforts to develop effective colorectal cancer methylation markers in cfDNA, so that early diagnosis of cancer and cancer risk is possible. It is unexpectedly found that specific regions of SLIT2 and PAX5 genes are methylated in colorectal cancer cells, and the specific regions of these genes are used as methylation markers to complete the present application.

[0069] In the present application, the inventors have made a long-term exploration and a large number of clinical sample verifications, and unexpectedly found that the methylation levels of SLIT2 and PAX5 genes are significantly different between colorectal cancer and non-colorectal cancer. SLIT2 is one of the members of the axon guidance factor ligand family, and its physiological functions are numerous and complex, and it plays an indispensable role in biological development, neurogenesis, tumor progression, etc.; PAX5 gene encodes a member of the paired box (PAX) family of transcription factors. The core feature of this gene family is a novel, highly conserved DNA-binding motif, the paired box. Paired box transcription factors are important regulators of early development, and changes in their gene expression are thought to contribute to tumor transformation. This gene encodes a B-cell lineage-specific activator protein that is expressed at early but not late stages of B-cell differentiation.

[0070] Amplification curve is a graph that detects product accumulation during polymerase chain reaction (PCR). It is generated by monitoring the increase in fluorescence signal in the reaction solution. The following are the characteristics of a typical PCR amplification curve:

[0071] Initial phase:

[0072] Threshold Cycle (Ct) value: In the early stage of PCR reaction, the fluorescence signal may be low, but as the PCR product accumulates, the fluorescence signal gradually increases. Ct value refers to the number of cycles required in the PCR reaction to make the fluorescence signal rise above the threshold value set in advance. The lower the Ct value, the higher the initial amount of target DNA in the sample.

[0073] Exponential growth phase:

[0074] Exponential phase: In the middle of PCR reaction, PCR product grows exponentially. At this time, the increase in Ct value will be accelerated, reflecting the exponential growth of target DNA in PCR reaction.

[0075] Plateau phase:

[0076] Plateau phase: In the late stage of PCR reaction, the accumulation of PCR product reaches saturation, and no longer grows exponentially. The PCR amplification curve of this stage forms a platform, and the increase of Ct value becomes slow.

[0077] In some embodiments of the present application, the diagnosis is early diagnosis, in particular, the early stage is stage 0-I or stage II of colorectal cancer (CRC).

[0078] The solutions of the present application will be explained below with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained commercially.

[0079] Example 1 Discovery of colorectal cancer-specific methylation gene sites

[0080] In order to screen biomarkers specifically methylated in colorectal cancer, 548 blood samples of patients clinically diagnosed as colorectal cancer (also referred to as colorectal cancer positive samples) and 389 blood samples of patients clinically diagnosed as negative for colorectal cancer were collected in this example.

[0081] 1. Preparation of methylation DNA sample library

[0082] (1) DNA extraction

[0083] The extraction of cfDNA was performed according to the instructions of the commercial extraction kit.

[0084] The concentration and fragment distribution of nucleic acids were controlled by Qubit 4.0 and Qsep 100, respectively. The yield of cfDNA extracted from 2 mL of human plasma should be greater than 5 ng, and cfDNA should have an enrichment peak at 167 bp or nearby. When the yield is greater than 50 ng, Qsep 100 capillary electrophoresis is used for fragment quality control, and when there is large fragment contamination, magnetic beads are used for fragment screening to remove large fragments.

[0085] (2) Library construction

[0086] The kit can be constructed with commercial library according to the method indicated in the instruction. The end repair, "A" tailing, and adapter ligation can be performed by the same type of kit such as Rapid Plus DNA LibPrep Kit for illumina (Cat. No. RK20208, ABclonal) or VAHTS Universal Pro DNA Library Prep Kit for illumina (Cat. No. ND608-02, Vazyme).

[0087] (3) Methylation DNA immunoprecipitation

[0088] Each methylation enrichment reaction can complete 12-100 cfDNA library mixtures (about 10 ng of each cfDNA sample library is input), and 10-24 genomic DNA library mixtures (about 100 ng of each genomic DNA library is input). The methylation enrichment based on 5-methylcytosine (5mC) antibody principle can be operated according to the method indicated in the instruction of the commercial methylation enrichment kit or self-prepared reagent. In this embodiment, zymo MeDIP kit (item number D5101-A) is used. Then, the methylation enrichment reaction is purified according to the instruction, and 10-12 rounds of conventional PCR amplification are performed with universal sequencing primer pairs to obtain a methylation DNA fragment library with a yield of more than 500 ng per reaction.

[0089] 2. Preparation of DNA probe library

[0090] (1) Probe design

[0091] The inventors selected the longest SLIT2 and PAX5 gene transcripts in the NCBI database to confirm the gene location. The sequence information in the promoter, 5'UTR, first exon (exon 1), and 1 kb upstream of the start codon was screened.

[0092] The obtained SLIT2 and PAX5 gene target methylation regions were designed for probes. The design principles of the probes are as follows: (1) full coverage of the target region without gaps;

[0093] (2) no overlap;

[0094] (3) each probe is 120 nt long.

[0095] The target region of the specific genome corresponding position and the probe coverage region of the specific genome corresponding position designed therefrom are shown in Table 1.

[0096] (2) Probe synthesis

[0097] According to the above probe design principle, 50 probes were designed for the methylation region of SLIT2 and PAX5 genes, covering all possible CpG sites. The probe coverage information is shown in Table 1.

[0098] Table 1, probe coverage information

[0099]

[0100]

[0101] 3. DNA capture probe hybridization

[0102] Liquid hybridization capture was performed using NadPrep hybridization capture reagent (Cat. No. REF1005101, NanoDynamics). Hybridization capture reaction can be single or multiple. The total amount of MeDIP amplification library input for each hybridization capture reaction should be between 300 ng and 8 μg. After purification, 500 ng of library (less than 500 ng was all input), Human Cot DNA and Nad Nano Blockers were added to the vacuum concentrator preheated to 42°C, and dried at a rotation speed of 1000 rpm. After drying, the prepared hybridization reaction solution (containing the above probe panel) was added, and oscillated and centrifuged. The hybridization program was 95°C / 30 sec; 65°C / Hold (100°C hot cover) for 4-16 hours. Then, the washed streptavidin magnetic beads were added to the hybridization system and incubated for 40 minutes, with vortex mixing every 10 minutes to ensure complete resuspension of the magnetic beads. It is worth noting that the reaction temperature of hybridization capture is 65°C, which is different from the 63°C of the methylation probe designed based on bisulfite conversion.

[0103] After the completion of the hybridization capture reaction, the combined magnetic beads were washed with four kinds of washing solution provided in the kit. Each step required discarding the residual liquid. Finally, 20 μL of nuclease-free water was added and gently vortexed.

[0104] 4. PCR amplification and purification after hybridization capture

[0105] PCR amplification of the product after hybrid capture, reagents using VAHTS Universal Pro DNA LibraryPrep Kit for illumina (Cat. No. ND608-02, Vazyme) in the amplification reagent for the experiment, the number of cycles in 12-13. After amplification, the same volume of VAHTS DNA Clean Beads (Cat. No. N411-03, Vazyme) is used to purify the product, and a relatively pure hybrid capture library is obtained; Qubit 4.0 is used for library concentration quantitative determination, and Qsep100 full-automatic nucleic acid protein analyzer is used for library fragment size detection.

[0106] 5. Library sequencing and bioinformatics analysis

[0107] The library concentration needs to be diluted to 4nM, mixed according to the required data amount, and the total data amount should not exceed 120G. After mixing, 5μL library is taken out, 5μL 0.2N NaOH is added, and it is mixed by blowing, denatured for 5 minutes, and then 990μL of HT1 Buffer (REF: 15058251, illumina) is immediately added after the end, vortexed, 105μL is taken out and added to 1295μL of HT1 Buffer, vortexed, and then the library for sequencing is obtained, with a concentration of 1.5pM.

[0108] The sequencer is NextSeq 550Dx produced by illumina, the reagents are High Output Reagent Cartridge v2 (REF: 15057929, illumina) (300 cycles), High Output Flow Cell Cartridge v2.5 (REF: 20022408, illumina) and Buffer Cartridge v2 (REF: 15057941, illumina), 1300μL of sequencing library is added to the sample site of High Output Reagent Cartridge v2, and each reagent is placed in turn, and the sequencing can start; this example uses double-end sequencing, and the total length is about 30 hours.

[0109] 6. Quality control of sequencing data

[0110] The raw data was quality controlled using Fastp (version 0.22.0) to remove low-quality bases, and the clean data had a whole Q20 of more than 90% and a Q30 of more than 85%. The average sequencing depth was about 300x. The average on-target rate of the probes in the above-mentioned probe combination was more than 80%, which showed that the combination of methylation immunoprecipitation and liquid-phase hybridization capture probes based on the present embodiment was feasible and effective for detecting cancer-related methylation regions.

[0111] 7. Analysis of colorectal cancer-related SLIT2 and PAX5 gene methylation difference regions

[0112] The DiffBind tool (version 3.8.4) was used to screen for differential peaks of tumors and non-tumors, and two algorithms, DESeq and EdgeR, were used to perform intersection and prioritize the regions within the panel. The screening criteria were: 1) False Discovery Rate (FDR) <0.01, and 2) Fold change <-1. The most significant characteristic methylation regions of the colorectal cancer positive group and the colorectal cancer negative group that met the conditions were selected as methylation difference regions.

[0113] In the present embodiment, the most significant characteristic methylation regions of the colorectal cancer positive group and the colorectal cancer negative group were selected from the regions of the SLIT2 and PAX5 genes covered by the 50 probes as methylation difference regions. The methylation CpG sites of these methylation difference regions are shown in Table 2.

[0114] Table 2: Methylation CpG sites of methylation difference regions

[0115]

[0116]

[0117] The RPM (Reads per million mapped reads) of the methylation difference regions (reads region covering the CpG sites in Table 2) in 548 colorectal cancer positive samples and 389 colorectal cancer negative samples was analyzed, and the P value was less than 0.005, which was significantly different.

[0118] Example 2: Detection of SLIT2 and PAX5 methylation genes in clinical samples and comparison of different treatment schemes

[0119] To further verify the clinical performance of differentially methylated regions of the SLIT2 and PAX5 genes associated with colorectal cancer in colorectal cancer plasma samples, the inventors used qPCR to test two additional groups of samples (62 plasma samples clinically diagnosed with CRC and 60 plasma control samples negative for CRC by colonoscopy). Among the 62 plasma samples clinically diagnosed with CRC, there were 14 samples of CRC stage 0-I, 15 samples of CRC stage II, 15 samples of CRC stage III, and 18 samples of CRC stage IV.

[0120] The flowchart for SLIT2 and PAX5 methylation gene detection is as follows: Figure 1 As shown, specifically:

[0121] (1) DNA extraction

[0122] cfDNA was extracted using a commercially available extraction kit, following the instructions in the manufacturer's manual.

[0123] Quality control of nucleic acid concentration and fragment distribution was performed using Qubit 4.0 and Qsep100, respectively. The yield of cfDNA extracted from 4 mL of human plasma should be greater than 10 ng, with an enrichment peak at or near 167 bp. When the yield exceeds 50 ng, Qsep100 capillary electrophoresis is used for fragmentation quality control. For large fragment contamination, magnetic beads are used for fragment screening to remove large fragments.

[0124] (2) Treatment of methylated DNA

[0125] ①Methylated DNA Immunoprecipitation

[0126] Take half of the total extracted nucleic acid and perform cfDNA methylation enrichment. Different reactions are carried out. Methylation enrichment based on the 5mC antibody principle is performed using the zymoMeDIP kit (catalog number D5101-A). After the methylation enrichment reaction is carried out according to the instructions, the elution volume is 50 μL.

[0127] ②Methylated DNA bisulfite conversion

[0128] Half of the total extracted nucleic acid was subjected to cfDNA methylation and bisulfite treatment. This was carried out in different reactions. Based on the principle of bisulfite conversion, the methylated DNA treatment was performed using the ZYMO RESEARCH DNA Transformation Kit (EZ DNA Methylation Kit, D5002). The elution volume was 50 μL.

[0129] (3) qPCR detection

[0130] The primer probe was synthesized by Shanghai Bailingge Biotechnology Co., Ltd., and the specific sequence information is as follows:

[0131] The sequence of the methylation DNA immunoprecipitation enrichment Taqman MGB probe primer pair is shown in Table 3.

[0132] Table 3, methylation DNA immunoprecipitation enrichment Taqman MGB probe primer pair

[0133]

[0134] The 3' end of the probe is labeled with MGB, the 5' end is labeled with a fluorescent group (FAM), and the 3' end is labeled with a quenching group (BHQ1).

[0135] Using the methylation DNA immunoprecipitation enrichment as a template, PCR amplification was performed, and the final concentration of each primer was 10 μM. Each gene was amplified by single reaction. The PCR reaction system was 5 μL of enriched template DNA, 2.5 μL of premix containing the above primers; PCR reaction reagent (2×Rapid Taq Master Mix) 17.5 μL, and the total volume was supplemented with water to 35 μL. The PCR reaction conditions are as follows: 95℃ for 5 minutes, 95℃ for 15 seconds, 60℃ for 40 seconds, and 45 cycles of amplification.

[0136] The sequence of the Taqman MGB probe primer pair after bisulfite conversion is shown in Table 4.

[0137] Table 4, Taqman MGB probe primer pair after bisulfite conversion

[0138]

[0139] The 3' end of the probe is labeled with MGB, the 5' end is labeled with a fluorescent group (FAM), and the 3' end is labeled with a quenching group (BHQ1).

[0140] Using the DNA after bisulfite conversion as a template, PCR amplification was performed, and the final concentration of each primer was 10 μM. Each gene was amplified by single reaction. The PCR reaction system was 5 μL of enriched template DNA, 2.5 μL of premix containing the above primers; PCR reaction reagent (2×Rapid Taq Master Mix) 17.5 μL, and the total volume was supplemented with water to 35 μL. The PCR reaction conditions are as follows: 95℃ for 5 minutes, 95℃ for 15 seconds, 60℃ for 40 seconds, and 45 cycles of amplification.

[0141] (4) Analysis of clinical sample detection results

[0142] The data were analyzed, and 122 samples were detected by methylation DNA immunoprecipitation enrichment and bisulfite DNA treatment and qPCR.

[0143] Figures 2A-2D The sample of the same CRC patient (sample number: CRC104435) was detected by methylation DNA immunoprecipitation enrichment and bisulfite DNA treatment, and the qPCR amplification curve was shown, wherein Figure 2A and 2B SLIT2 and PAX5 gene methylation DNA immunoprecipitation enrichment qPCR amplification curves, Ct values were 33.14 and 35.98, respectively, Figure 2C and 2D bisulfite DNA qPCR amplification curves, Ct values were 38.23 and 38.03, respectively, and the methylation DNA immunoprecipitation enrichment qPCR detection had obvious advantages.

[0144] The Ct values of the samples with Ct values > 45 or undetermined Ct values were set to 45, and the logistic regression formula was used for calculation, and the ROC curves were drawn according to the calculation results. As shown in Figure 3A and Figure 3B The areas under the ROC curves based on the two different methods were 0.969 and 0.958, respectively. According to the ROC curve, the threshold (cut-off value) was set for different methods: the cut-off value of the methylation DNA immunoprecipitation enrichment qPCR detection (i.e., methylation DNA immunoprecipitation fluorescence quantitative PCR method) was set to logistic scores = 800; the cut-off value of the bisulfite conversion qPCR detection (i.e., bisulfite conversion fluorescence quantitative PCR method) was set to logistic scores = 725. If the amplification logistic scores values of the two genes SLIT2 and PAX5 of the detected sample are equal to or lower than the set cut-off value, the sample is determined to be negative, otherwise it is determined to be positive. Thus, the detection results of 122 samples were statistically analyzed. The formula is as follows:

[0145] Formula I: logistic scores = e k / (1+e k )

[0146] Methylation DNA immunoprecipitation (Formula II): k = -2.478 x Ct SLIT2 -3.076 x Ct PAX5 +203.157 bisulfite conversion (Formula III): k = -0.36 x CtSLIT2 -1.373 x Ct PAX5 +67.241

[0147] Table 5 shows the comparison of the qPCR detection based on the methylation DNA immunoprecipitation enrichment method with the colonoscopy results (gold standard), Table 6 shows the comparison of the qPCR detection based on the bisulfite conversion treatment with the colonoscopy results, and Table 7 shows the comparison of the detection results of the qPCR detection based on the methylation DNA immunoprecipitation enrichment method and the qPCR detection based on the bisulfite conversion treatment.

[0148] Table 5, Comparison of qPCR detection results based on the methylation DNA immunoprecipitation enrichment method with colonoscopy results

[0149]

[0150] Table 6, Comparison of qPCR detection results based on the bisulfite conversion treatment with colonoscopy results

[0151]

[0152] Table 7, Comparison of the qPCR detection based on the methylation DNA immunoprecipitation enrichment method with the qPCR detection based on the bisulfite conversion treatment

[0153]

[0154] As shown in Tables 5-7, the SLIT2 and PAX5 gene methylation difference regions have higher sensitivity (95.2%) to CRC when verified by the qPCR detection platform based on the methylation DNA immunoprecipitation enrichment method, and also maintain high specificity (95.0%) to non-intestinal cancer samples, with an accuracy of 95.1%. The overall performance is better than the qPCR detection based on the bisulfite conversion treatment.

[0155] (5) Analysis of detection results of clinical colorectal cancer staging samples

[0156] Among the above-mentioned 37 plasma samples clinically diagnosed as CRC, there were 14 samples of CRC 0-I stage, 15 samples of CRC II stage, 15 samples of CRC III stage, and 18 samples of CRC IV stage. The qPCR detection based on the methylation DNA immunoprecipitation enrichment method was compared with the qPCR detection based on the bisulfite conversion treatment, and the detection and statistical analysis of different CRC pathological staging samples are shown in Tables 8-9:

[0157] Table 8, Detection of different CRC pathological staging samples

[0158]

[0159] Table 9, sensitivity statistics analysis of different CRC pathological stages

[0160]

[0161] From Tables 8-9, it can be seen that the methylation difference regions of SLIT2 and PAX5 genes maintain high sensitivity (92.9%, 93.3%) in the verification of the methylation DNA immunoprecipitation enrichment method qPCR detection platform for CRC 0-I and II stage samples, and the performance for early detection of CRC is better than that of the bisulfite conversion treatment qPCR detection, providing a new potential marker for early detection of CRC. Since the present application maintains high sensitivity in the early detection of CRC, it can be used for the prediction of CRC. The present application uses cfDNA as the analysis sample, which is easy to obtain, and since cfDNA contains a lot of information, it is not limited to the diagnosis (including auxiliary diagnosis) and / or prediction of CRC, but can also be used for the prediction and / or diagnosis of other diseases, and has the characteristics of being superior to single tissue samples in the context of high-throughput sequencing analysis.

[0162] The principles and implementation modes of the present application are described herein by applying specific examples, and the above examples are only used to help understand the method of the present application and its core idea; at the same time, for those skilled in the art, according to the idea of the present application, changes will be made in the specific implementation mode and application range, and changes and improvements of the present application will be possible without exceeding the concept and scope defined by the claims, and the content of the above-mentioned embodiments of the specification should not be understood as a limitation of the present application.

Claims

1. Use of a combination of methylation molecular markers for detecting diagnosis or prognosis of colorectal cancer in the manufacture of a product for diagnosing or prognosing colorectal cancer, characterized in that, The methylation molecular marker combination for diagnosing or predicting colorectal cancer consists of the following two methylation molecular markers located in human SLIT2 and PAX5 genes respectively: chr4:20253208-20253408 and chr9:37002603-37002803, which are specifically located by hg38; The reagent for detecting the methylation molecular marker combination for diagnosing or predicting colorectal cancer comprises primer and probe combination a and / or primer and probe combination b; Primer and probe combination a: a primer pair as shown in SEQ ID No. 1 and SEQ ID No. 2 and a Taqman MGB probe as shown in SEQ ID No. 3 for fluorescence quantitative PCR amplification of chr4:20253208-20253408; a primer pair as shown in SEQ ID No. 4 and SEQ ID No. 5 and a Taqman MGB probe as shown in SEQ ID No. 6 for fluorescence quantitative PCR amplification of chr9:37002603-37002803; Primer and probe combination b: a primer pair as shown in SEQ ID No. 7 and SEQ ID No. 8 and a Taqman MGB probe as shown in SEQ ID No. 9 for fluorescence quantitative PCR amplification of chr4:20253208-20253408; a primer pair as shown in SEQ ID No. 10 and SEQ ID No. 11 and a Taqman MGB probe as shown in SEQ ID No. 12 for fluorescence quantitative PCR amplification of chr9:37002603-37002803.

2. Use according to claim 1, wherein The reagent for detecting the methylation molecular marker combination for diagnosing or predicting colorectal cancer comprises any one or more reagents used in the following methods, which comprise at least one of methylation DNA immunoprecipitation fluorescence quantitative PCR method, bisulfite conversion fluorescence quantitative PCR method, bisulfite conversion sequencing method, methylation chip sequencing, methylation-specific PCR method.

3. A kit for diagnosis or prognosis of colorectal cancer, characterized by, The kit is used for detecting the methylation molecular marker combination; the methylation molecular marker combination consists of the following two methylation molecular markers located in human SLIT2 and PAX5 genes respectively: chr4:20253208-20253408 and chr9:37002603-37002803, which are specifically located by hg38; The kit comprises primer and probe combination a and / or primer and probe combination b; Primer and probe combination a: primer pair as shown in SEQ ID No. 1 and SEQ ID No. 2 and Taqman MGB probe as shown in SEQ ID No. 3 for the fluorescent quantitative PCR amplification of chr4:20253208-20253408; primer pair as shown in SEQ ID No. 4 and SEQ ID No. 5 and Taqman MGB probe as shown in SEQ ID No. 6 for the fluorescent quantitative PCR amplification of chr9:37002603-37002803; Primer and probe combination b: primer pair as shown in SEQ ID No. 7 and SEQ ID No. 8 and Taqman MGB probe as shown in SEQ ID No. 9 for the fluorescent quantitative PCR amplification of chr4:20253208-20253408; primer pair as shown in SEQ ID No. 10 and SEQ ID No. 11 and Taqman MGB probe as shown in SEQ ID No. 12 for the fluorescent quantitative PCR amplification of chr9:37002603-37002803.

4. The kit of claim 3, wherein Also included are one or more of the required reagents for the Rapid Taq Master Mix, methylation enrichment based on the principle of 5-methylcytosine antibody, and methylation conversion based on the principle of bisulfite conversion.

5. The kit of claim 3, wherein The 3' end of the Taqman MGB probe is provided with a MGB and a fluorescence quenching group, and the 5' end is provided with a fluorescence group; the combination formed by the fluorescence quenching group and the fluorescence group is selected from BHQ1 or NFQ and FAM, BHQ2 and VIC or HEX, BHQ2 and Cy3, and BHQ2 and Cy5.

6. A computer-readable storage medium, characterized in that, The program can be executed by the processor to realize the analysis and processing of the fluorescent quantitative PCR detection data of the methylation molecular marker combination for the diagnosis or prediction of colorectal cancer, to obtain a colorectal cancer determination result, including the following steps: Ct obtained by performing the fluorescent quantitative PCR under the condition of primer and probe combination a using the DNA enriched by methylated DNA immunoprecipitation cfDNA as a template SLIT2 Ct PAX5 , and the detection result is processed and judged by using formula I and formula II; Formula I: logistic scores = e k / (1 + e k ) Equation II: k = -2.478 x Ct SLIT2 -3.076 x Ct PAX5 +203.157 A logistic scores score ≤800 is determined to be negative for colorectal cancer, and a logistic scores score >800 is determined to be positive for colorectal cancer; If the Ct value obtained from the fluorescent quantitative PCR detection result is >45 or no Ct value is detected, the Ct value is all taken as 45 to be substituted into the formula II; Primer and probe combination a: primer pair as shown in SEQ ID No. 1 and SEQ ID No. 2 and Taqman MGB probe as shown in SEQ ID No. 3 for the fluorescent quantitative PCR amplification of chr4:20253208-20253408 to obtain Ct SLIT2 ; primer pair as shown in SEQ ID No. 4 and SEQ ID No. 5 and Taqman MGB probe as shown in SEQ ID No. 6 for the fluorescent quantitative PCR amplification of chr9:37002603-37002803 to obtain Ct PAX5 ; The methylation molecular marker combination for the diagnosis or prediction of colorectal cancer is composed of the following two methylation molecular markers located at human SLIT2 and PAX5 genes: chr4:20253208-20253408 and chr9:37002603-37002803, which are specifically located by hg38.

7. A computer readable storage medium characterized in that, The program can be executed by the processor to realize the analysis and processing of the fluorescent quantitative PCR detection data of the methylation molecular marker combination for the diagnosis or prediction of colorectal cancer, to obtain a colorectal cancer determination result, including the following steps: Ct obtained by performing fluorescent quantitative PCR under the condition of primer and probe combination b using the DNA converted by the bisulfite as a template SLIT2 Ct PAX5 , and the detection result is processed and judged by using formula I and formula III; Formula I: logistic scores = e k / (1 + e k ) Equation III: k = -0.36 x Ct SLIT2 -1.373 x Ct PAX5 +67.241 The colorectal cancer is determined as negative when the logistic scores score is ≤725, and as positive when the logistic scores score is >725; The Ct value obtained from the result of the fluorescent quantitative PCR detection is >45 or the Ct value is not detected, and the Ct value is all taken as 45 to be substituted into the formula III; Primer and probe combination b: primer pair as shown in SEQ ID No. 7 and SEQ ID No. 8 and Taqman MGB probe as shown in SEQ ID No. 9 for the fluorescent quantitative PCR amplification of chr4:20253208-20253408 to obtain Ct SLIT2 ; primer pair as shown in SEQ ID No. 10 and SEQ ID No. 11 and Taqman MGB probe as shown in SEQ ID No. 12 for the fluorescent quantitative PCR amplification of chr9:37002603-37002803 to obtain Ct PAX5 ; the methylation molecular marker combination for the diagnosis or prognosis of colorectal cancer consists of the following 2 methylation molecular markers located at the human SLIT2 and PAX5 genes: chr4:20253208-20253408 and chr9:37002603-37002803, specific localization by hg38.

8. The computer-readable storage medium of claim 6 or 7, wherein, The fluorescent quantitative PCR detection data analyzed and processed by the program come from the following fluorescent quantitative PCR reaction: the reaction system is 35 μL; the reaction liquid reagent used is 2×Rapid Taq Master Mix; and the reaction condition is 95℃ for 5 minutes, 95℃ for 15 seconds, 60℃ for 40 seconds, and 45 cycles of amplification.

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