Primer probe group, kit, detection method and application for alimentary canal tumor marker detection

A digestive tract tumor, primer probe technology, applied in biochemical equipment and methods, microbial measurement/testing, recombinant DNA technology, etc., can solve the limitations, cannot be used for early screening of digestive tract malignant tumors, time-consuming and labor-intensive And other issues

Pending Publication Date: 2020-08-21
SUZHOU VERSABIO TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 group, kit, detection method and application for alimentary canal tumor marker detection
  • Primer probe group, kit, detection method and application for alimentary canal tumor marker detection
  • Primer probe group, kit, detection method and application for alimentary canal tumor marker detection

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Experimental program
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Effect test

Embodiment 1

[0034] 10000, 1000, 100 and 10 copies / reaction KCNQ5 gene methylation-positive nucleic acid solutions after bisulfite conversion were used as detection objects. Select the primer probe groups of SEQ NO.2, SEQ NO.3, SEQ NO.4, SEQ NO.5, SEQ NO.6, and SEQ NO.7, and the fluorescent quantitative PCR reaction system is: the primer concentration is 0.4mM, the probe Concentration 0.2mM, 1XPCR buffer, 6mMMgCl 2 Solution, 0.1U / ul DNA polymerase, PCR reaction mixing volume is 15ul, DNA template volume is 15ul, reaction conditions are 95°C for 20 minutes, (95°C for 10 seconds, 56°C for 30 seconds, 72°C for 10 seconds) X 50 cycles , 40°C for 1 minute.

[0035] The result is as figure 1 As shown, the primer probe set and kit of the present invention have good reproducibility and high sensitivity for detection of 10000, 1000, 100 and 10 copies / reaction KCNQ5 gene methylation-positive nucleic acid solutions.

Embodiment 2

[0037] With 6 pairs of colorectal cancer and adjacent tissues and 4 pairs of gastric cancer and adjacent tissues as detection objects, the DNA was extracted with the QIAGEND Neasy Blood&Tissue kit, and transformed with the bisulfite rapid transformation kit of Suzhou Weishan Biotechnology Co., Ltd. and purification. Select the primer probe groups of SEQ NO.2, SEQ NO.3, SEQ NO.4, SEQ NO.5, SEQ NO.6, and SEQ NO.7, and the fluorescent quantitative PCR reaction system is: primer concentration 0.2mM, probe concentration 0.1Mm, 1.5XPCR buffer, 6mM MgCl 2 Solution, 0.12U / ul DNA polymerase, PCR reaction mixing volume is 15ul, DNA template volume is 15ul, reaction conditions are 95°C for 20 minutes, (95°C for 10 seconds, 56°C for 30 seconds, 72°C for 10 seconds) X 50 cycles , 40°C for 30 seconds. The result analysis was compared with the KCNQ5 gene Ct value minus the ACTB gene Ct value after data normalization.

[0038] Test results such as figure 2 As shown, the methylation level...

Embodiment 3

[0040] The feces of 7 cases of advanced adenoma, 23 cases of colorectal cancer and 32 cases of normal people were used as detection objects.

[0041] After the DNA was extracted with the Fecal Human Genomic DNA Extraction Kit from Suzhou Weishan Biotechnology Co., Ltd., the DNA was transformed and purified using the bisulfite rapid conversion kit from Suzhou Weishan Biotechnology Co., Ltd. Choose SEQNO.2, SEQ NO.3, SEQ NO.4, SEQ NO.5, SEQ NO.6, SEQ NO.7 primer-probe group, fluorescent quantitative PCR reaction system is: primer concentration is 0.4mM, probe concentration 0.2mM, 1XPCR buffer, 6mM MgCl 2 Solution, 0.12U / ul DNA polymerase, PCR reaction mixing volume is 15ul, DNA template volume is 15ul, reaction conditions are 95°C for 20 minutes, (95°C for 10 seconds, 56°C for 30 seconds, 72°C for 10 seconds) X 50 cycles , 40°C for 1 minute. The test results are shown in Table 1 and image 3 shown.

[0042] Table 1. The kit of the present invention detects the results of col...

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Abstract

The invention discloses a primer probe group, a kit, a detection method and application for alimentary canal tumor marker detection, and belongs to the technical field of biological medicines. A disclosed marker is a methylated KCNQ5 gene and has at least one modified CpG locus, and the detection method comprises the following steps: extracting DNA in a human-derived biological sample; convertingthe extracted DNA through methylated bisulfite, and converting the unmethylated cytosine C into uracil U; carrying out methylation real-time fluorescent quantitative PCR reaction by adopting the kit;and interpreting according to the Ct value of the KCNQ5 gene, and judging whether the subject suffers from digestive tract tumor or not. The kit is used for detecting specific DNA methylation markersin biological samples of high-risk groups and patients with malignant tumors of digestive tracts, and a high-sensitivity and non-invasive detection means can be provided for early screening, early diagnosis and personalized treatment of malignant tumors of digestive tracts.

Description

technical field [0001] The invention relates to a primer probe set, a kit, a detection method 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 com...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2600/166C12Q2600/158C12Q2523/125C12Q2531/113C12Q2563/107
Inventor 赵国栋熊尚岷
Owner SUZHOU VERSABIO TECH INC
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