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Digestive tract tumor marker combination, detection kit and application thereof

A technology of digestive tract tumor and detection kit, applied in the field of biomedicine, can solve the problems of low acceptance, limitation, inability to use early screening of digestive tract malignant tumors, etc., and achieve the effect of low missed detection rate

Active Publication Date: 2021-04-13
SUZHOU VERSABIO TECH INC
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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
In China, there are nearly 800 million people (>40 years old) who need early screening of the digestive tract, and less than 5% of the people who have actually participated in early screening, which means early diagnosis and early treatment of digestive tract tumors in China still face great challenges

Method used

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  • Digestive tract tumor marker combination, detection kit and application thereof
  • Digestive tract tumor marker combination, detection kit and application thereof
  • Digestive tract tumor marker combination, detection kit and application thereof

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Experimental program
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Effect test

Embodiment 1

[0053] Taking the methylation-positive genomic DNA solution after bisulfite conversion as the detection object, set 4 concentration extractions, 10, 100, 1000, 10000 copies / reaction. Use SEQ ID 1-3, SEQ ID 7-12, SEQ ID 31-33 to prepare KCNQ5, C9orf50, CLIP4 and ACTB mixed detection fluorescent quantitative PCR reaction system, the fluorescent quantitative PCR reaction system is: the primer concentration is 0.4mM, the 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, 58°C for 30 seconds, 72°C for 10 seconds) × 50 Cycle, 40°C for 1 minute.

[0054] The result is as Figure 1-3 As shown, methylated KCNQ5, C9orf50, and CLIP4 can all be well amplified, and the minimum reaction concentration can reach 10 copies / reaction.

Embodiment 2

[0056] Taking the methylation-positive genomic DNA solution after bisulfite conversion as the detection object, set 4 concentration extractions, 10, 100, 1000, 10000 copies / reaction. Use SEQ ID 16-18, SEQ ID 22-23, SEQ ID 28-33 to prepare ZNF582, TFPI2, ELMO1 and ACTB mixed detection fluorescence quantitative PCR reaction system, the fluorescence quantitative PCR reaction system is: primer concentration is 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, 58°C for 30 seconds, 72°C for 10 seconds) × 50 Cycle, 40°C for 1 minute.

[0057] The result is as Figure 4-6 As shown, methylated ZNF582, TFPI2, and ELMO1 can all be well amplified, and the minimum reaction concentration can reach 10 copies / reaction.

Embodiment 3

[0059] Taking the blood of 10 cases of esophageal cancer, 54 cases of gastric cancer, 34 cases of colorectal cancer and 67 normal control samples as the test object, 10 mL of blood was drawn respectively and 3.5 mL of plasma was separated, using the free nucleic acid extraction kit of Suzhou Weishan Biotechnology Co., Ltd. After the cfDNA was extracted, it was converted and purified using the bisulfite rapid conversion kit from Suzhou Weishan Biotechnology Co., Ltd. Use SEQ ID 1-7, SEQ ID 13-15, SEQ ID 31-33 to prepare KCNQ5, C9orf50, CLIP4 and ACTB mixed detection fluorescent quantitative PCR reaction system 1, use SEQ ID 19-21, SEQ ID 25-33 to prepare ZNF582, Fluorescent quantitative PCR reaction system 2 for mixed detection of TFPI2, ELMO1 and ACTB; the remaining components of the above two groups of fluorescent quantitative PCR reaction systems are: primer concentration 0.4mM, probe concentration 0.1mM, 1×PCR buffer, 6mM MgCl 2 Solution, 0.12U / ul DNA polymerase, PCR reacti...

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Abstract

The invention belongs to the field of biomedicine, and particularly relates to a digestive tract tumor marker combination, a detection kit and application thereof. Marker genes comprise methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO1; and the sequences of the methylated KCNQ5, C9orf50, CLIP4, ZNF582, TFPI2 and ELMO1 are shown as SEQ ID NO.34-SEQ ID NO.39 respectively. According to the invention, a non-invasive method capable of simultaneously detecting esophageal cancer, gastric cancer and colorectal cancer, a marker composition and a use method thereof are developed, and a new technology for early prevention and control of digestive tract tumors is provided.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a digestive tract tumor marker combination, a detection kit and applications thereof. Background technique [0002] According to the latest statistics from the World Health Organization (WHO) on the incidence and mortality of malignant tumors in the world, in 2018, colorectal cancer, gastric cancer and esophageal cancer accounted for 12%, 11% and 7% of the total number of malignant tumors in China, respectively. The combined incidence of the above three cancers accounts for 30% of the total number of malignant tumors, ranking first among all types of cancer. In terms of mortality, gastric cancer accounts for 14%, esophageal cancer accounts for 10%, and colorectal cancer accounts for 9%, ranking second, fourth, and fifth in the incidence and mortality of malignant tumors in China, and the combined deaths of the three cancers The rate reaches 33%, also ranking first among al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2600/166C12Q2531/113C12Q2545/113C12Q2523/125Y02A50/30
Inventor 赵国栋熊尚岷王凯
Owner SUZHOU VERSABIO TECH INC
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