Primer probe set and reagent kit for detecting digestive tract tumor marker and application of reagent kit

A technology of digestive tract tumors and primer probes, which is applied in the determination/testing of microorganisms, recombinant DNA technology, biochemical equipment and methods, etc. Realize issues such as endoscopic screening

Pending Publication Date: 2020-10-20
SUZHOU VERSABIO TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many deficiencies in the endoscopic detection method: 1. Due to the traditional concept and people's lack of awareness of the benefits of endoscopic screening, the awareness of early diagnosis and early treatment is not strong, and the discomfort caused by endoscopic examination exists. Great concerns, so the willingness of residents to participate in endoscopic screening is not high; 2. At present, the free endoscopic screening project led by the government has only been tried out in a small number of high-risk groups in areas with a high incidence of digestive tract malignancies in my country, and most areas Endoscopic screening is not included in the scope of government medical insurance payment, and my country is still a developing country. Even in rural areas of developed provinces like Jiangsu, endoscopic screening is still a relatively high cost for ordinary families. Limiting the motivation of residents to actively seek endoscopy screening
3. Endoscopic screening requires many conditions such as experienced endoscopists, expensive endoscopic equipment, and complete endoscopic cleaning and disinfection facilities. Therefore, in a country with a large population like my country, due to the lack of necessary medical and health resources and The human resources of endoscopists cannot achieve convenient endoscopic screening, especially in areas with relatively backward economic development.
At the same time, endoscopic examinations in most hospitals need to be booked in advance, which is time-consuming and labor-intensive, which also limits endoscopic examinations as the first-line screening method for digestive tract malignancies
4. As an invasive screening method, endoscopic screening needs to be prepared in advance, such as taking laxatives or anesthetics, and it is very painful during the testing process. For Chinese people with strong traditional concepts, it violates privacy, so very low acceptance
However, some other existing protein tumor markers such as CEA and CA199 can only detect a small number of advanced cancers, and the detection rate of early cancers and cancers is less than 30%, which cannot be used for early screening of digestive tract malignancies

Method used

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  • Primer probe set and reagent kit for detecting digestive tract tumor marker and application of reagent kit
  • Primer probe set and reagent kit for detecting digestive tract tumor marker and application of reagent kit
  • Primer probe set and reagent kit for detecting digestive tract tumor marker and application of reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The detection object is colorectal advanced adenoma, colorectal cancer and gastric cancer genomic DNA solution after bisulfite conversion. Prepare the fluorescent quantitative PCR reaction system with primer probe set 1, the fluorescent quantitative PCR reaction system is: primer concentration 0.4mM, probe concentration 0.1mM, 1×PCR buffer, 6mM MgCl 2 Solution, 0.1U / ul DNA polymerase, PCR reaction mixing volume is 15ul, DNA template volume is 15ul, reaction condition is 95°C for 20 minutes, (95°C for 10 seconds, 56°C for 30 seconds, 72°C for 10 seconds) × 50 Cycle, 40°C for 1 minute.

[0040] The result is as figure 1 As shown, wherein, 1 is colorectal cancer, 2 is colorectal advanced adenoma, and 3 is gastric cancer. Genomic DNA has an obvious amplification curve and is positive.

Embodiment 2

[0042] Using 5 pairs of colorectal cancer and para-cancerous tissues as detection objects, DNA was extracted with QIAGEN DNeasy Blood&Tissue Kit, and transformed and purified with Bisulfite Rapid Conversion Kit from Suzhou Weishan Biotechnology Co., Ltd. Prepare the fluorescent quantitative PCR reaction system with primer probe set 2: primer concentration 0.2mM, probe concentration 0.1Mm, 1×PCR buffer, 6mM MgCl 2 Solution, 0.12U / ul DNA polymerase, PCR reaction mixing volume is 15ul, DNA template volume is 15ul, reaction condition is 95°C for 20 minutes, (95°C for 10 seconds, 60°C for 30 seconds, 72°C for 15 seconds) × 50 Cycle, 40°C for 30 seconds.

[0043] Test results such as figure 2As shown, the methylation level of CLIP4 gene in colorectal cancer was significantly higher than that in paracancerous tissues, proving that methylated CLIP4 gene can be used to distinguish colorectal cancer from cancer tissues and non-cancer tissues.

Embodiment 3

[0045] Using 5 pairs of colorectal cancer and para-cancerous tissues as detection objects, DNA was extracted with QIAGEN DNeasy Blood&Tissue Kit, and transformed and purified with Bisulfite Rapid Conversion Kit from Suzhou Weishan Biotechnology Co., Ltd. Prepare the fluorescent quantitative PCR reaction system with primer probe set 3: primer concentration 0.4mM, probe concentration 0.1Mm, 1.5×PCR buffer, 6mM MgCl 2 Solution, 0.12U / ul DNA polymerase, PCR reaction mixing volume is 15ul, DNA template volume is 15ul, reaction condition is 95°C for 20 minutes, (95°C for 10 seconds, 60°C for 30 seconds, 72°C for 15 seconds) × 50 Cycle, 40°C for 30 seconds.

[0046] Test results such as image 3 As shown, the methylation level of CLIP4 gene in colorectal cancer was significantly higher than that in paracancerous tissues, proving that methylated CLIP4 gene can be used to distinguish colorectal cancer from cancer tissues and non-cancer tissues.

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Abstract

The invention discloses a digestive tract tumor marker. The maker is a methylated CLIP4 gene, and has one or more modified CpG sites. The invention further discloses a primer probe set and reagent kitfor detecting the digestive tract tumor marker. The sequence of the primer probe set comprises one or more base sites being in complementary pairing with the modified CPG sites. The digestive tract tumor maker is used for detecting a CLIP4 gene of the special DNA methylation marker in biology samples of digestive tract malignant tumor high-risk groups and patients, and a high sensitivity non-invasive detection means is provided for early screening, early diagnosis and individualized treatment for digestive tract malignant tumors.

Description

technical field [0001] The invention relates to a primer probe group, a kit and an application thereof for detecting digestive tract tumor markers, and belongs to the technical field of biomedicine. Background technique [0002] According to the latest data on the incidence and mortality of malignant tumors released by the World Health Organization (WHO), there were 4,285,033 new cases of malignant tumors and 2,865,174 deaths from malignant tumors in China in 2018. Among them, malignant tumors of the digestive tract (colorectal cancer, gastric cancer, liver cancer, esophageal cancer, and pancreatic cancer) rank among the top ten in terms of morbidity and mortality among all malignant tumors. The morbidity and mortality of colorectal cancer account for more than 50% of the global morbidity and mortality, and the incidence of colorectal cancer is also showing a gradual upward trend in China. Therefore, malignant tumors of the digestive tract are the most common type of malign...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/6886C12Q1/686C12Q2600/154C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 赵国栋
Owner SUZHOU VERSABIO TECH INC
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