CN114507738A - Methylation site, application of product for detecting methylation level and kit - Google Patents
Methylation site, application of product for detecting methylation level and kit Download PDFInfo
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- CN114507738A CN114507738A CN202210301745.6A CN202210301745A CN114507738A CN 114507738 A CN114507738 A CN 114507738A CN 202210301745 A CN202210301745 A CN 202210301745A CN 114507738 A CN114507738 A CN 114507738A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
The application belongs to the technical field of medical detection, and relates to a methylation site, application of a product for detecting methylation level and a kit. The methylation level of cg11909912CpG sites of MAPT genes in a PANC1 cell line is obviously higher than that of a normal control, the CpG sites can be used as a pancreatic cancer diagnosis marker, the approach of cancer clinical detection is expanded, and the screened specific sites can be effectively used for rapid diagnosis of pancreatic cancer. Compared with traditional biopsy, CT, MRI and other imaging examinations, the method has the advantages of no damage to human body, high accuracy and repeatability.
Description
Technical Field
The application relates to a methylation site, application of a product for detecting methylation level and a kit, and belongs to the technical field of medical detection.
Background
The DNA methylation spectrum comprehensively and systematically shows the dynamic change of the related immunity of the liver cancer, and has prominent advantages in the research of early diagnosis markers of the liver cancer: the body's immune system functions through "immune surveillance", including: antigen recognition, antigen presentation, cytotoxic action and the like are involved in the whole process of tumor occurrence and development, and the involved immune cells and immune molecules are hundreds of in number and present dynamic network changes, and single monitoring of certain cells or cytokines is not enough to reflect the whole change of the immune system of the organism in the occurrence and development of liver cancer. Therefore, although it is well known that body immunity plays an important role in controlling the occurrence and development of liver cancer, no immune marker has been found to be useful for diagnosing liver cancer. With the completion of human genome sequencing programs and the wide application of genomics, proteomics and high-throughput sequencing technologies, the important role of DNA methylation in the aspects of normal cell development, heredity, gene expression regulation, gene repair, chromosome stability and the like is also highlighted. The DNA methylation spectrum covering 450000 sites of immune function can comprehensively and systematically show the subtle changes of the immune system in the disease progression of liver cancer at various stages. Therefore, dynamic changes of liver cancer related immunity are comprehensively shown through DNA methylation spectrums, and then liver cancer early diagnosis markers are screened to become new liver cancer diagnosis markers for research.
In the prior art, there are various methods for monitoring DNA methylation. For example, Bisulfite Sequencing PCR (BSP) is a method developed by Frommer and colleagues in 1992 for the study of DNA methylation, and is one of the most commonly used techniques in the field of methylation research. BSP combines bisulfite treatment of genomic DNA (conversion of unmethylated cytosine bases to uracil) and PCR amplification of regions of interest within the DNA. The amplification products are then routinely sequenced to assess the level of DNA methylation within the DNA sequence of interest. The method is characterized in that methylated cytosine does not react with sodium bisulfite, so that the purpose of distinguishing methylated or unmethylated CpG dinucleotides can be achieved through routine sequencing.
However, at present, there is no uniform and accurate standard for detecting whether methylation is abnormal, and the risk of cancer cannot be predicted as early as possible by the change of methylation level at a specific site.
Disclosure of Invention
The application aims to provide a methylation site, application of a product for detecting methylation level and a kit, which can be used for diagnosing pancreatic cancer by detecting a specific methylation site of a MAPT gene.
In order to achieve the purpose, the application provides the following technical scheme:
in a first aspect, the present application provides the use of a methylation site of a MAPT gene, cg11909912, as a diagnostic marker for pancreatic cancer.
In a second aspect, the application also provides a use of the product for detecting the methylation level of the MAPT gene in a sample to be detected, and the product is used for preparing a diagnostic article for pancreatic cancer.
As one example, the product detects the methylation level of the MAPT gene in the test sample by any one or more of pyrosequencing, bisulfite sequencing, quantitative and/or qualitative methylation specific polymerase chain reaction, quantitative and/or qualitative bisulfite specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing in combination with bisulfite, southern blotting, restriction landmark genomic scanning, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, in combination with sodium bisulfite restriction endonuclease analysis, or mass spectrometry.
As one example, the product detects the region from-523 bp upstream to 146bp downstream of the MAPT gene.
As one example, the product detects the cg11909912 site of the MAPT gene.
As one example, the product comprises a primer for detecting the cg11909912 locus, and the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.
As one example, the product uses bisulfite sequencing to detect the methylation level of MAPT genes in the test sample.
As one example, the sample to be tested is whole blood or a blood sample, and the blood sample is serum or plasma.
In a third aspect, the application provides a qualitative detection kit for methylation of MAPT genes, which comprises a primer for detecting cg11909912 locus of the MAPT genes, and the sequences of the primer are shown as SEQ ID NO.1 and SEQ ID NO. 2.
In one embodiment, the kit further comprises any one of an enzyme mixture, a dNTP mixture, and a PCR buffer, or a combination of at least two of them.
Compared with the prior art, the beneficial effect of this application lies in: the methylation level of cg11909912CpG sites of MAPT genes in a PANC1 cell line is obviously higher than that of a normal control, the CpG sites can be used as a pancreatic cancer diagnosis marker, the approach of cancer clinical detection is expanded, and the screened specific sites can be effectively used for rapid diagnosis of pancreatic cancer. Compared with traditional biopsy, CT, MRI and other imaging examinations, the method has the advantages of no damage to human body, high accuracy and repeatability.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a schematic illustration of an MAPT amplification sequence shown in an embodiment of the present application; the total length of the amplification sequence is 138bp, 10 sites are covered, the underlined part is a probe sequence, and the No. 5 CpG site is a CpG site detected by the probe, namely a differential methylation gene site;
FIG. 2 is a CG island prediction plot of CpG sites of a MAPT gene probe according to an embodiment of the present application; the black square area is a predicted CG island;
FIG. 3 is a graph of MAPT monoclonal methylation patterns of the cell line PANC1 and normal human samples, as shown in one example of the present application; the arrows indicate the differentially methylated gene sites.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.
It should be noted that: in this application, "accuracy" refers to the degree to which a measured or calculated quantity (test report value) corresponds to its actual (or true) value. Clinical accuracy refers to the ratio of true outputs (true positive (TP) or True Negative (TN) to misclassified outputs (false positive (FP) or False Negative (FN)), and can be expressed as sensitivity, specificity, Positive Predictive Value (PPV) or Negative Predictive Value (NPV), Matheus Correlation Coefficient (MCC), or likelihood, odds ratio, Receiver Operating Characteristic (ROC) curve, Area Under Curve (AUC), among other measures.
A "formula," "algorithm," or "model" is any mathematical formula, algorithm, analytical or stylistic process, or statistical technique that takes one or more continuous or categorical inputs (referred to herein as "parameters") and calculates an output value (sometimes referred to as an "index" or "exponent value"). Non-limiting examples of "formulas" include sums, ratios, and regression operators (e.g., coefficients or powers (exponents)), biomarker value conversion and normalization (including but not limited to those normalization schemes based on clinical-determinants such as gender, age, or race), rules and criteria, statistical classification models, and neural nets trained on historical populations. Linear and non-linear formulas and statistical classification analysis are used among the combined determinants to determine the relationship between the level of the detected determinant in a subject sample and the probability of a subject having an infection or a certain type of infection. Of particular interest in group and combinatorial construction are structural and syntactic statistical classification algorithms, and methods of exponential construction, using pattern recognition features, including established techniques such as cross-correlation, Principal Component Analysis (PCA), factor rotation, logistic regression (LogReg), Linear Discriminant Analysis (LDA), Eigenene Linear Discriminant Analysis (ELDA), Support Vector Machine (SVM), Random Forest (RF), recursive partitioning Tree (RPART), and other related decision tree classification techniques, reduced center of gravity (SC), SteIC, Kth-Nearest Neighbor relation, Boosting, decision trees, Neural Networks, Bayesian Networks, and hidden Markov models, to name a few. Other techniques may be used in the survival and time of event hazard analysis, including Cox, Weibull, Kaplan-Meier, and Greenwood models, which are well known to those skilled in the art. Many of these techniques can be used in combination with determinant selection techniques, such as forward selection, backward selection, or stepwise selection, comprehensive investigation of all potential groups of a given size, genetic algorithms, or biomarker selection methods that may themselves be included within their own technology. These can be combined with information criteria such as Akaike Information Criteria (AIC) or Bayes Information Criteria (BIC) to quantify the trade-off between other biomarkers and model improvement and to help minimize overfitting. The resulting predictive model can be validated in other studies, or cross validated in initially trained studies, using techniques such as Bootstrap, Leave-One-out (LOO), and 10-Fold cross validation (10-Fold CV). In various steps, the false discovery rate may be estimated by value permutation according to techniques known in the art.
For the diagnostic (or prognostic) interventions of the present invention, since each output (which may entail different costs for TP, FP, TN, or FN in a disease classification diagnostic test), the health economic utility function may be based on clinical and individual output costs and values, preferably tending towards sensitivity exceeding specificity, or PPV exceeding NPV, thus providing another measure of health economic performance and value, which may be different from the more direct clinical or analytical performance measure. These different measures and relative tradeoffs will generally converge only in the case of perfect tests with zero error rate (also called zero predicted object output misclassification or FP and FN), and all performance measures will tend to be imperfect, but to a different degree.
By "measuring", "determining", "detecting" or "examining" is meant assessing the presence, absence, quantity or amount (which can be an effective amount) of a given substance or subject-derived sample (comprising a derivative of a qualitative or quantitative concentration level of such substance) in a clinical setting, or otherwise assessing the value or classification of a subject's non-analyte clinical parameter or clinical-determinant.
A "sample" in the context of the present invention is a biological sample isolated from a subject and can include, for example, but is not limited to, whole blood, serum, plasma, saliva, mucus, respiratory air, urine, CSF, saliva, sweat, stool, hair, semen, biopsy, rhinorrhea, tissue biopsy, cytological sample, platelets, reticulocytes, leukocytes, epithelial cells, or whole blood cells.
By "statistically significant" is meant that the change is greater than would be expected by chance alone (may be a "false positive"). Statistical significance can be determined by any method known in the art. A commonly used measure of significance includes a p-value, which represents the probability that at least a threshold value will yield a result at a given data point, assuming that the data point is a single contingent result. The p value is 0.05 or less, and the results are generally considered to be highly significant.
The source of the sample in the context of the present invention is preferably a human. The subject may be male or female. A subject is a human that has been previously diagnosed or identified as having an infection, and optionally has undergone or is undergoing therapeutic intervention for the infection. Alternatively, the subject may also be a subject that has not been previously diagnosed with an infection. For example, the subject may display one or more risk factors for infection.
In this application, "methylation" refers to methylation of CpG sites.
The methylation level of cg11909912CpG sites of MAPT genes in a PANC1 cell line is obviously higher than that of a normal control, the CpG sites can be used as a pancreatic cancer diagnosis marker, the approach of cancer clinical detection is expanded, and the screened specific sites can be effectively used for rapid diagnosis of pancreatic cancer.
According to an exemplary embodiment of the present application, there is provided a use of a methylation site of a MAPT gene as a diagnostic marker for pancreatic cancer, the methylation site being cg 11909912.
According to an exemplary embodiment of the present application, there is provided a use of a product for detecting methylation level of MAPT gene in a test sample, for preparing a diagnostic product for pancreatic cancer.
Preferably, the product detects the methylation level of the MAPT gene in the sample to be tested by any one or more of pyrosequencing, bisulfite sequencing, quantitative and/or qualitative methylation specific polymerase chain reaction, quantitative and/or qualitative bisulfite specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing in combination with bisulfite, southern blotting, restriction landmark genomic scanning, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, in combination with sodium bisulfite restriction endonuclease analysis, or mass spectrometry.
Preferably, the product detects the region from upstream-523 bp to downstream 146bp of the MAPT gene.
Preferably, the product detects the cg11909912 site of the MAPT gene.
Preferably, the product comprises a primer for detecting the cg11909912 locus, and the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.
Preferably, the product detects the methylation level of the MAPT gene in the sample to be detected by a bisulfite sequencing method.
Preferably, the sample to be tested is whole blood or a blood sample, and the blood sample is serum or plasma.
According to an exemplary embodiment of the present application, the present application provides a qualitative detection kit for methylation of MAPT gene, which comprises a primer for detecting cg11909912 site of the MAPT gene, wherein the sequence of the primer is shown as SEQ ID No.1 and SEQ ID No. 2.
Preferably, the kit further comprises any one of an enzyme mixture, a dNTP mixture, and a PCR buffer, or a combination of at least two of them.
The present application will be described in further detail with reference to specific examples, wherein those skilled in the art and those who do not recognize specific techniques or conditions are referred to in the literature of the art or in the product specification. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.
Referring to fig. 1 and fig. 2, in this example, based on the MAPT gene bisulfite sequencing, the methylation states of 1 CpG site in a specific region (-523, 146) of the promoter region of the human MAPT gene were detected, and compared with that of PANC1 cell line, specifically:
primer probe design is carried out based on the cg11909912 site of the HM450K probe, and the upstream primer and the downstream primer thereof are respectively shown in SEQ ID No.1 and SEQ ID No. 2:
F:5’-TTGTTAATGGAGGAATTTTAGGGGGA-3’
R:5’-TCCRTACCCAAACATTTCCAA-3’。
the genome DNA of the PANC1 cell line is extracted by adopting a genome DNA extraction kit, and the genome DNA of a normal human whole blood sample (EDTA anticoagulation) is extracted by adopting a blood genome DNA extraction kit. After the PANC1 cell line and normal human gDNA were modified by the bisulfite conversion kit, PCR reactions (25. mu.L) were performed under the conditions shown in Table 1, wherein the PCR program is shown in Table 2.
TABLE 1 PCR reaction System
TABLE 2 PCR reaction procedure
Subsequently, a 2% agarose gel was prepared, and the PCR product was electrophoresed at a voltage of 120V for 30min using 100bp Ladder. The size of the product is 173bp, the gel imager is used for observation, the position of the target fragment is found, and the gel block is cut off and then the target fragment is purified and recovered by using the gel recovery kit.
The product was recovered to pGEM-T vector overnight at 4 ℃ under 1. mu.L of pGEM-T vector, 1. mu. L T4 ligase, 5. mu.L of 2 Xbuffer and 3. mu.L of gel recovery product. The TA cloning products were then all transformed into the T1 Simple cloning vector, plated in culture medium (Amp +), and incubated overnight in an incubator at 37 ℃. A plurality of monoclonal colonies were picked into 1mL LB medium (Amp +), cultured at 180rpm at 37 ℃ for 12h, sequenced and differential methylated gene site verified.
Table 3 shows MAPT monoclonal methylation rate analysis of the cell line PANC1 and normal human samples, and in combination with fig. 3, the results show that the methylation level of cg11909912CpG site in PANC1 cell line is significantly higher than that of normal control, and the selected specific site can be effectively used for rapid diagnosis of pancreatic cancer. Based on this, cg11909912CpG site methylation can be used as a diagnostic marker for pancreatic cancer.
TABLE 3 MAPT monoclonal methylation rate analysis of cell line PANC1 and normal human samples
In summary, the following steps: the methylation level of cg11909912CpG site of MAPT gene in PANC1 cell line is obviously higher than that of normal control, and the gene can be used as a pancreatic cancer diagnosis marker, thus expanding the approach of cancer clinical detection, and the screened specific site can be effectively used for rapid diagnosis of pancreatic cancer. Compared with traditional biopsy, CT, MRI and other imaging examinations, the method has the advantages of no damage to human body, high accuracy and repeatability.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
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Claims (10)
1. Use of a methylation site of a MAPT gene as a diagnostic marker for pancreatic cancer, wherein the methylation site is cg 11909912.
2. The application of a product for detecting the methylation level of MAPT genes in a sample to be detected is characterized in that the product is used for preparing a pancreatic cancer diagnosis product.
3. The use according to claim 2, wherein the product detects the level of methylation of MAPT genes in the test sample by a method selected from any one or more of pyrosequencing, bisulfite sequencing, quantitative and/or qualitative methylation specific polymerase chain reaction, quantitative and/or qualitative bisulfite specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing in combination with bisulfite, southern blotting, restriction landmark genomic scanning, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease analysis, or mass spectrometry.
4. The use of claim 2, wherein the product detects the region from-523 bp upstream to 146bp downstream of the MAPT gene.
5. The use of claim 4, wherein the product detects the cg11909912 site of the MAPT gene.
6. The use of claim 5, wherein the product comprises a primer for detecting the cg11909912 site, the primer having the sequence shown in SEQ ID No.1 and SEQ ID No. 2.
7. The use of claim 6, wherein said product is used to detect the level of methylation of MAPT genes in said test sample by bisulfite sequencing.
8. The use of claim 2, wherein the test sample is whole blood or a blood fraction, and the blood fraction is serum or plasma.
9. A MAPT gene methylation qualitative detection kit is characterized by comprising a primer for detecting cg11909912 locus of the MAPT gene, wherein the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.
10. The kit of claim 9, further comprising any one of an enzyme mixture, a dNTP mixture, or a PCR buffer, or a combination of at least two thereof.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114731987A (en) * | 2022-05-19 | 2022-07-12 | 苏州药诺泰克新药研发有限公司 | Mouse model for craniosynostosis, construction method and application thereof |
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| US20060068414A1 (en) * | 2004-07-26 | 2006-03-30 | Kennedy Brian K | Identification of aging genes through large-scale analysis |
| CN108300787A (en) * | 2018-04-17 | 2018-07-20 | 中国科学院北京基因组研究所 | Special application of the methylation sites as early diagnosing mammary cancer marker |
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| CN114731987A (en) * | 2022-05-19 | 2022-07-12 | 苏州药诺泰克新药研发有限公司 | Mouse model for craniosynostosis, construction method and application thereof |
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