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CN-121992105-A - Biomarker and detection kit for early diagnosis of pancreatic cancer

CN121992105ACN 121992105 ACN121992105 ACN 121992105ACN-121992105-A

Abstract

The invention discloses a biomarker and a detection kit for early diagnosis of pancreatic cancer, and belongs to the technical field of biological medicines. The invention discloses a method for preparing a biomarker for early diagnosis of pancreatic cancer, which is characterized in that CpG dinucleotide methylation sites in a MDGA gene promoter region CpG island can be used as a biomarker for early diagnosis of pancreatic cancer, fills the blank of research on association between MDGA gene and pancreatic cancer in the prior art, and has extremely high specificity and sensitivity. The marker targets a specific CpG island of MDGA gene shown in SEQ ID NO. 1, only shows a remarkable hypermethylation state in pancreatic cancer tissues, has extremely low methylation level in normal pancreatic tissues, can effectively distinguish pancreatic cancer tissues from normal pancreatic tissues, simultaneously avoids the defects that the traditional serum marker is easy to be interfered by benign diseases and has high false positive rate, and provides a totally new specific target for early diagnosis of pancreatic cancer.

Inventors

  • WANG ZHIXIN
  • ZHOU BO
  • ZHAO DANYUN
  • LIU YU

Assignees

  • 天津国际旅行卫生保健中心(天津海关口岸门诊部)

Dates

Publication Date
20260508
Application Date
20260409

Claims (10)

  1. 1. A biomarker for early diagnosis of pancreatic cancer, which is characterized in that the biomarker is a CpG dinucleotide methylation site in a CpG island of a promoter region of a gene MDGA, and the nucleotide sequence of the gene MDGA is shown as SEQ ID NO. 1.
  2. 2. The biomarker of claim 1, wherein the CpG island meets the conditions of a GC content of >55%, obsCpG/ExpCpG >0.65, and a length of greater than 500 bases.
  3. 3. A MDGA methylation detection kit for early diagnosis of pancreatic cancer, which is characterized by comprising a specific detection reagent for detecting the methylation level of a CpG island in the promoter region MDGA of the gene shown in SEQ ID No. 1.
  4. 4. The methylation detection kit of claim 3, wherein the CpG island meets the conditions of a GC content of >55%, obsCpG/ExpCpG >0.65, and a length of > 500 bases.
  5. 5. The methylation detection kit of claim 3, wherein the specific detection reagent comprises a methylation PCR reaction solution for detecting the degree of methylation of CpG dinucleotide sites of at least one target region in the promoter region of the MDGA gene.
  6. 6. The methylation detection kit of claim 5, wherein the methylation PCR reaction solution comprises two methylation specific primer pairs, one methylation primer pair comprises an upstream primer MDGA MF and a downstream primer MDGA MR, the sequence of the upstream primer MDGA MF is shown as SEQ ID NO. 6, the sequence of the downstream primer MDGA MR is shown as SEQ ID NO. 7, and the other methylation primer pair comprises an upstream primer MDGA UF and a downstream primer MDGA UR, the sequence of the upstream primer MDGA UF is shown as SEQ ID NO. 8, and the sequence of the downstream primer MDGA UR is shown as SEQ ID NO. 9.
  7. 7. The methylation detection kit of any one of claims 4 to 6, further comprising an auxiliary reagent required for a methylation PCR reaction, the auxiliary reagent required for a methylation PCR reaction comprising Taq hot start enzyme, 10 x methylation PCR specific buffer, dNTP mix, negative control, and positive control; The negative control is double distilled water without DNase, the positive control comprises a methylation positive control and a non-methylation positive control, the methylation positive control is a gene promoter region CpG island fragment shown in SEQ ID NO. 1 which is methylated in vitro, and the non-methylation positive control is a MDGA gene non-methylation DNA standard substance derived from normal pancreatic tissues.
  8. 8. The MDGA gene expression detection kit for early diagnosis of pancreatic cancer is characterized by comprising a PCR amplification reaction solution, wherein the PCR amplification reaction solution is used for detecting the mRNA expression level of a gene MDGA2 shown in SEQ ID NO. 1.
  9. 9. The gene expression test kit of claim 8, wherein the PCR amplification reaction solution comprises an expression specific primer pair consisting of an upstream primer MDGA F and a downstream primer MDGA R, wherein the sequence of the upstream primer MDGA F is shown as SEQ ID No. 2, and the sequence of the downstream primer MDGA R is shown as SEQ ID No. 3.
  10. 10. The gene expression test kit of claim 8 or 9, further comprising an auxiliary reagent required for a PCR reaction, the auxiliary reagent required for the PCR reaction comprising Taq hot start enzyme, 10 x PCR buffer, dNTP mix, negative control and positive control; wherein, the negative control is double distilled water, and the positive control is a corresponding methylation positive DNA or mRNA expression positive control sample.

Description

Biomarker and detection kit for early diagnosis of pancreatic cancer Technical Field The invention belongs to the technical field of biological medicines, and particularly relates to a biomarker and a detection kit for early diagnosis of pancreatic cancer. Background Pancreatic cancer is one of the tumors with the highest malignancy in the digestive system, the onset and progress of the pancreatic cancer are hidden, the clinical symptoms lack of specificity, most patients are in middle and late stages when they are diagnosed, the optimal treatment time is missed, the survival rate of the patients is extremely low in 5 years, and the human life and health are seriously threatened. Therefore, the early screening and early diagnosis of pancreatic cancer are realized, the survival rate of patients is improved, the critical rupture of prognosis is improved, and the important clinical requirements to be solved in the field are urgent. Currently, the means for diagnosis of pancreatic cancer in clinic mainly include imaging examinations, serum tumor marker detection and histopathological examinations. The imaging examination (such as ultrasound, CT, MRI and the like) has low sensitivity to early pancreatic cancer detection, is difficult to find micro focus, is easily influenced by factors such as the operation level of an inspector, focus position and the like, cannot meet the requirement of large-scale early screening, and the histopathological examination is used as a diagnosis gold standard, belongs to invasive examination, is difficult to be used for early screening and routine monitoring of high-risk groups, and is only suitable for the diagnosis of clinical suspected cases. The serum tumor marker detection is a common means for early tumor screening due to the characteristics of simple operation, noninvasive property, repeatability and the like, and the pancreatic cancer related serum markers commonly used clinically at present mainly comprise saccharide antigen 19-9 (CA 19-9), saccharide antigen 242 (CA 242), carcinoembryonic antigen (CEA) and the like. However, these markers have defects of insufficient specificity and sensitivity, have low positive detection rate in early stages of pancreatic cancer, are easily interfered by benign diseases such as biliary tract diseases and pancreatitis, have false positive results, cannot effectively distinguish pancreatic cancer from benign pancreatic diseases, and are difficult to be used as reliable indexes for early diagnosis of pancreatic cancer. With the development of epigenetic and molecular biological technologies, gene methylation and abnormal gene expression have been demonstrated to be important molecular events in the process of tumorigenesis and development, wherein CpG island methylation in a gene promoter region can lead to gene transcriptional silencing, thereby regulating and controlling the expression of tumor-associated genes and affecting tumorigenesis and development. Compared with the traditional serum markers, the gene methylation markers and the gene expression markers have the advantages of strong specificity, high sensitivity, capability of generating abnormality in early disease and the like, become research hotspots in the early diagnosis field of tumors, and methylation or abnormal expression of a plurality of genes are tried to be used for early diagnosis of tumors such as lung cancer, stomach cancer and the like, but in the field of pancreatic cancer, a core marker with both high specificity and high sensitivity is not found. The MDGA (MAM Domain Containing Glycosylphosphatidylinositol Anchor) gene is a gene encoding glycosyl phosphatidyl inositol anchoring protein, the existing research only reports that the gene is related to nervous system development, no document or patent discloses the relevance of MDGA gene and pancreatic cancer, no methylation site of MDGA gene promoter region CpG island is found to be used as a biomarker for early diagnosis of pancreatic cancer, and no special detection kit aiming at the methylation site and MDGA gene expression level exists. In summary, the existing diagnostic markers and detection methods have obvious defects due to the lack of efficient, accurate and noninvasive early diagnosis means of pancreatic cancer in clinic at present, and cannot meet the requirements of early clinical screening and diagnosis. Therefore, the pancreatic cancer early diagnosis biomarker with strong specificity and high sensitivity is developed, a corresponding special detection kit is developed, the early accurate diagnosis of pancreatic cancer is realized, and the pancreatic cancer early diagnosis biomarker has important clinical value and application prospect and is a technical problem to be solved urgently by the technicians in the field. Disclosure of Invention Aiming at the technical defects of poor specificity, low sensitivity, lack of special markers, detection products and the like of the existing early pancreatic c