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CN-116103402-B - Esophageal cancer diagnosis kit based on polygene methylation level detection and application thereof

CN116103402BCN 116103402 BCN116103402 BCN 116103402BCN-116103402-B

Abstract

The invention provides an esophageal cancer diagnosis kit based on polygene methylation level detection, which is used for realizing early screening of esophageal cancer samples by detecting methylation conditions of esophageal cancer gene methylation detection sites including ZNF154, RNF126, C2ORF27A, OTOP, TM4SF19 and P16, and has the advantages of absolute quantification, high batch stability and high sensitivity based on the combination of the kit and digital PCR, can accurately distinguish the rule between the gene methylation of the samples and the esophageal cancer, adopts a fluorescent marker probe for typing detection, has strong specificity and is suitable for esophageal cancer patients in different periods, thereby providing a convenient, quick and accurate detection means for early screening, diagnosis and prognosis of esophageal cancer, and having important clinical value.

Inventors

  • GAO MINGHANG

Assignees

  • 易瑞达(福建)生物技术有限公司

Dates

Publication Date
20260505
Application Date
20230104

Claims (7)

  1. 1. The esophageal cancer diagnosis kit based on polygene methylation level detection is characterized in that esophageal cancer gene methylation detection sites detected by the kit comprise ZNF154, RNF126, C2ORF27A, OTOP2, TM4SF19 and P16, the sequences of which are shown as SEQ ID NO.1-12, and the kit comprises a characteristic detection reagent, wherein the characteristic detection reagent comprises dye quantitative PCR premix and the following primer and probe combination: 1) The primer and probe combination for ZNF154 methylation detection comprises a Mut specific primer shown as SEQ ID NO.13, a Mut specific primer shown as SEQ ID NO.14, a Mut specific probe shown as SEQ ID NO.25, a Wt specific primer shown as SEQ ID NO.31, a Wt specific primer shown as SEQ ID NO.32 and a Wt specific probe shown as SEQ ID NO. 43; 2) Primer and probe combinations for RNF126 methylation detection, which comprise a Mut-specific primer shown as SEQ ID NO.15, a Mut-specific primer shown as SEQ ID NO.16, a Mut-specific probe shown as SEQ ID NO.26, a Wt-specific primer shown as SEQ ID NO.33, a Wt-specific primer shown as SEQ ID NO.34, and a Wt-specific probe shown as SEQ ID NO. 44; 3) The primer and probe combination for detecting the methylation of the C2ORF27A comprises a Mut specific primer shown as SEQ ID NO.17, a Mut specific primer shown as SEQ ID NO.18, a Mut specific probe shown as SEQ ID NO.27, a Wt specific primer shown as SEQ ID NO.35, a Wt specific primer shown as SEQ ID NO.36 and a Wt specific probe shown as SEQ ID NO. 45; 4) The primer and probe combination for OTOP < 2 > methylation detection comprises a Mut specific primer shown as SEQ ID NO.19, a Mut specific primer shown as SEQ ID NO.20, a Mut specific probe shown as SEQ ID NO.28, a Wt specific primer shown as SEQ ID NO.37, a Wt specific primer shown as SEQ ID NO.38 and a Wt specific probe shown as SEQ ID NO. 46; 5) The primer and probe combination for detecting the methylation of the TM4SF19 comprises a Mut specific primer shown as SEQ ID NO.21, a Mut specific primer shown as SEQ ID NO.22, a Mut specific probe shown as SEQ ID NO.29, a Wt specific primer shown as SEQ ID NO.39, a Wt specific primer shown as SEQ ID NO.40 and a Wt specific probe shown as SEQ ID NO. 47; 6) The primer and probe combination for P16 methylation detection comprises a Mut specific primer shown as SEQ ID NO.23, a Mut specific primer shown as SEQ ID NO.24, a Mut specific probe shown as SEQ ID NO.30, a Wt specific primer shown as SEQ ID NO.41, a Wt specific primer shown as SEQ ID NO.42 and a Wt specific probe shown as SEQ ID NO. 48.
  2. 2. The esophageal cancer diagnosis kit based on polygenic methylation level detection according to claim 1, wherein the detection sample type of the kit is an in vitro blood sample.
  3. 3. The kit for diagnosing esophageal cancer based on the detection of the methylation level of multiple genes of claim 1, wherein the 5' -end of each specific probe comprises a fluorescent group, and the fluorescent groups comprise FAM and VIC.
  4. 4. The kit for diagnosing esophageal cancer based on polygenic methylation level detection as claimed in claim 3, wherein the fluorescent group of the Mut specific probe is FAM, and the fluorescent group of the Wt specific probe is VIC.
  5. 5. The esophageal cancer diagnostic kit based on polygenic methylation level detection according to claim 1, wherein the 3' -end of the specific probe comprises a quenching group, and the quenching group is MGB.
  6. 6. The esophageal cancer diagnosis kit based on the polygenic methylation level detection according to claim 1, wherein the reaction system of the kit comprises 1 mu L of treated DNA, namely a vulcanized template, 2.4 mu L of upstream and downstream primers with the concentration of 10 mu M, 0.75 mu L of Mut/Wt probe with the concentration of 10 mu M, 15 mu L of dye quantitative PCR premix and 8.45 mu L of water which are mixed into a uniform 30 mu L system.
  7. 7. The esophageal cancer diagnosis kit based on polygenic methylation level detection according to claim 1, wherein the PCR reaction conditions of the kit are as follows: 。

Description

Esophageal cancer diagnosis kit based on polygene methylation level detection and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to an esophageal cancer diagnosis kit based on polygene methylation level detection and application thereof. Background Esophageal cancer (Esophageal cancer, EC), a malignant tumor of the digestive tract, is the sixth leading cause of cancer-related death worldwide. Wherein the risk of Chinese morbidity is extremely high, and the world first of the new morbidity department (Scopus Preview-SCHILSKY R L A, et al NEJM 2020;383: 897-00). In general, patients with esophageal cancer only show different degrees of dysphagia in the early and middle stages, and the best time of treatment is very easy to miss. Until eating disorders appear, esophageal tumors of most patients have progressed into advanced stages, severely affecting clinical treatment and prognosis. Therefore, the early screening of the esophageal cancer is very critical, and the effective and convenient inspection method is beneficial to improving the cure rate and the life quality of patients with the esophageal cancer. The related detection method of the esophageal cancer mainly comprises tumor marker detection, esophageal cancer function detection, imaging and endoscopy detection, cell or histopathological detection and the like. Where endoscopy is good for early high sensitivity detection, extensive endoscopic screening is difficult because of the relatively expensive equipment, difficult handling and low patient acceptance (Codipilly D C, et al, gastroiintest Endosc. 2018; 88:413-426). The tumor markers are derived from the development process of tumors, can be collected from ex-vivo blood, saliva, excrement, epidermal cells and the like, have absolute detection advantages in various tumors, but still lack strong specific markers of esophageal cancer at present. Under the background of increasingly mature biotechnology, accurate medical treatment becomes an epoch-making call. In order to achieve the goal of early screening and diagnosing esophageal cancer for the crowd without obvious symptoms, the detection of nucleic acid molecules can become a reasonable choice. Like other tumors, various genetic or epigenetic changes cause changes in gene function, which are the direct cause of lesions and initiation of tumors in esophageal cells. Therefore, researchers are actively and widely exploring risk information (Zang B, et al, aging 2020; 12:3771-3790) contained in genes in esophageal cancer cases, and genetic control conditions of non-coding RNAs such as lncRNA（Liu H, et al. Pathol Oncol Res. 2020;26:1029-1039）、circRNA（Shoda K, et al. Biomedicines. 2022;10:1643）, and then providing information for diagnosis, prognosis and predictive treatment response of esophageal cancer to clinicians. Among them, gene methylation is a major form of epigenetic gene and has a very close relationship with canceration. The risk of cancer herein includes that hypermethylation of promoter regions can silence cancer suppressor genes and extensive genomic hypomethylation promotes activation of oncogenes and transformation of cells. Gene methylation has become a very important detection target in other tumor early sieves. Therefore, the deep research of methylation of the esophageal cancer genes is also helpful for promoting the solution of the early diagnosis problem of esophageal cancer, but the methylation gene combination application of the related detection of esophageal cancer is not found at home and abroad, so that the research and development of the methylation combination application-based esophageal cancer detection method with the characteristics of convenient implementation, rapidness, high sensitivity and specificity is urgently needed by the industry staff. Disclosure of Invention The invention aims to solve the technical problem of providing an esophageal cancer diagnosis kit based on polygene methylation level detection, which has the characteristics of high sensitivity, specificity and convenience in operation, is combined with digital PCR (polymerase chain reaction) and is suitable for esophageal cancer patients in different periods, thereby providing a convenient, quick and accurate detection means for early screening, diagnosis and prognosis of esophageal cancer, and having important clinical value. The invention solves the technical problems through the following technical scheme: An esophageal cancer diagnosis kit based on polygene methylation level detection, wherein the esophageal cancer gene methylation detection sites detected by the kit comprise ZNF154, RNF126, C2ORF27A, OTOP2, TM4SF19 and P16, the sequences of which are shown as SEQ ID NO.1-12, and the kit comprises a characteristic detection reagent, wherein the characteristic detection reagent comprises dye quantitative PCR premix and the following primer and probe combination: 1) The primer and