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Multiple lung cancer-related gene methylation combined detection kit, multiple lung cancer-related gene methylation combined detection method, and applications of multiple lung cancer-related gene methylation combined detection kit

A detection reagent and joint detection technology, which is applied in the field of multiple lung cancer-related gene methylation joint detection kits, can solve the problems of difficult early detection and early qualitative diagnosis, and difficult early diagnosis of lung cancer, so as to improve the detection rate and design Ingenious, simple structure effect

Pending Publication Date: 2018-05-01
上海透景诊断科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Early lung cancer is hardly noticed by doctors and patients because it often has no special symptoms, and it is difficult to detect and diagnose early with conventional diagnostic methods. In addition, some tumor markers can only be used as a preliminary screening or auxiliary diagnosis of lung cancer, but cannot be diagnosed. Therefore, early diagnosis of lung cancer is difficult

Method used

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  • Multiple lung cancer-related gene methylation combined detection kit, multiple lung cancer-related gene methylation combined detection method, and applications of multiple lung cancer-related gene methylation combined detection kit
  • Multiple lung cancer-related gene methylation combined detection kit, multiple lung cancer-related gene methylation combined detection method, and applications of multiple lung cancer-related gene methylation combined detection kit
  • Multiple lung cancer-related gene methylation combined detection kit, multiple lung cancer-related gene methylation combined detection method, and applications of multiple lung cancer-related gene methylation combined detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1, the selection of detection target gene

[0067] Methylated DNA has obvious advantages as a detection target. Compared with protein markers, DNA can be amplified and easily detected; compared with mutation markers, methylated DNA is located in a specific part of the gene , generally in the promoter region, making detection easier and more convenient. In the previous study, the methylated DNA detection results of different genes had a certain synergistic effect. In order to further improve the clinical positive detection rate, on the original basis (p16, H-cadherin (CDH13), SHOX2, HOXA9, RARβ, RASSF1A) were added The new ANKRD18B and MPDZ genes are used as candidate detection genes to further study the distribution of methylation sites of each gene, and design primers and probes for detection respectively. The detection primers and probes for each gene are as follows:

[0068] The detection primers and probes for p16 are:

[0069] pl6 Primer F: ACGTCGTGAG...

Embodiment 2

[0126] Embodiment 2, primer and probe design

[0127] In order to optimize the detection reagents suitable for simultaneous detection of human SHOX2 gene, human RASSF1A gene, human ANKRD18B gene and human MPDZ gene methylated DNA, the inventors conducted in-depth research on each gene sequence, after repeated research and comparison, selected The sequence of the amplified region of each gene is shown in Table 2.

[0128] Table 2

[0129]

[0130]

[0131]

[0132]

[0133] According to the regions determined in Table 2, the inventors further optimally designed specific primers and detection probes. The designed primers and detection probes are shown in Table 3. All primers and probes were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0134] table 3

[0135]

[0136]

Embodiment 3

[0137] Embodiment 3, different primer probe combination experiments

[0138] DNA extraction, sulfite modification, detection system preparation, and PCR amplification procedures were the same as in Example 1, and the amplification objects were modified sample DNA and control DNA (DNA fragments of SHOX2, RASS1FA, ANKRD18B, and MPDZ). After the experiment runs, the analysis steps are as follows:

[0139] (1) Determine whether the experiment is credible:

[0140] (a) There is signal in CY5, and the Ct value of CY5 signal is ≥12, the experiment is credible;

[0141] If the Ct value is less than 12, it indicates that the added DNA is too much, and the DNA should be diluted before doing it;

[0142] (b) If there is no CY5 signal, it indicates that the added DNA contains PCR inhibitors or the DNA treatment fails, and the DNA needs to be re-extracted and treated with sulfite;

[0143] Among them, the positive quality control product reaction tubes should have signals of FAM, VIC an...

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Abstract

The invention provides a multiple lung cancer-related gene methylation combined detection kit, which comprises: a detection reagent for specifically detecting the DNA methylation of human SHOX2 gene;a detection reagent for specifically detecting the DNA methylation of human RASSF1A gene; a detection reagent for specifically detecting the DNA methylation of human ANKRD18B gene; and a detection reagent for specifically detecting the DNA methylation of human MPDZ gene, wherein preferably the detection reagents are primers and probes. The invention further provides applications of the multiple lung cancer-related gene methylation combined detection kit in multiple lung cancer-related gene methylation combined detection, and a multiple lung cancer-related gene methylation combined detection method. According to the present invention, the multiple lung cancer-related gene methylation combined detection kit can perform the combined detection on the multiple lung cancer-related gene methylation, can significantly improve the tumor detection rate, has advantages of clever design and simple structure, and is suitable for large-scale promotion and applications.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to the technical field of gene methylation detection, and specifically refers to a multiple lung cancer-related gene methylation joint detection kit, joint detection method and application. Background technique [0002] Lung cancer has become one of the main causes of human cancer death. Lung cancer is the cancer with the highest incidence rate in China, and its morbidity and mortality are increasing rapidly. The incidence and mortality of lung cancer are the highest among all tumors, but lung cancer is not the most diagnosed tumor. In the United States, breast tumors and prostate tumors have a higher diagnosis rate, because early diagnosis and early treatment can be achieved, greatly Improve the 5-year survival rate (89 and 99%, respectively), while the 5-year survival rate of lung cancer is only 15%. [0003] In clinical practice, early diagnosis of lung cancer has always...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/154C12Q2600/16C12Q2600/166
Inventor 王方金陈静文郭安亮姚见儿
Owner 上海透景诊断科技有限公司
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