Fecal detection kit for advanced adenoid tumor and colorectal cancer related gene methylation locus

A detection kit and technology for colorectal cancer, applied in the medical field, can solve problems such as reduced sensitivity, difficulty in purchasing, and difficulty in large-scale use, and achieve high sensitivity and specificity, improved sensitivity, and low cost

Active Publication Date: 2019-03-19
杭州艾维克生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This technology also has corresponding disadvantages that make it difficult to use in large quantities
1. This kit is an imported reagent, which is relatively difficult to purchase
2. This kit is aimed at the American population, and the sensitivity of detection in the Chinese population is reduced
3. The specificity of this kit is relatively low, and a large number of "false positives" will be generated, causing panic in the subjects
[0009] There are still many shortcomings in the currently commercially available stool sample methylation detection kits for colorectal cancer
The detection sensitivity and specificity are difficult to meet the function of replacing or close to replacing sigmoidoscopy; there are technical difficulties in the preservation, extraction and bisulfite conversion of stool samples; the detection content of the detection kit is confusing and there is no clear research gene information, etc. ; The detection cost of the kit remains high, and the clinical application value is reduced, etc.

Method used

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  • Fecal detection kit for advanced adenoid tumor and colorectal cancer related gene methylation locus
  • Fecal detection kit for advanced adenoid tumor and colorectal cancer related gene methylation locus
  • Fecal detection kit for advanced adenoid tumor and colorectal cancer related gene methylation locus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] The present invention uses a nucleic acid extraction kit (product of Xiamen Aide Biomedical Technology Co., Ltd., article number: ADx-FF01 c1806264) to extract DNA from colorectal cancer tissue samples, advanced adenoma tissue samples, and paracancerous tissue samples. The extraction steps are as follows:

[0076] 1. According to the size of the tissue area of ​​the FFPE section, scrape 1 to 5 pieces of FFPE samples into a 1.5mL centrifuge tube, add 1mL xylene, shake and mix for 10s;

[0077] 2. Centrifuge at 13000×g for 2 minutes at room temperature, carefully remove the supernatant (do not suck the precipitate);

[0078] 3. Add 1mL of absolute ethanol, shake and mix for 10s, centrifuge at 13000×g for 2min at room temperature, carefully remove the supernatant (do not suck the precipitate);

[0079] 4. Leave the lid open for 10 minutes at room temperature or 5 minutes at 37°C to allow the ethanol to fully evaporate.

[0080] 5. Add 180 μL Buffer DTL and 20 μL Proteina...

Embodiment 2

[0139] Primer probes were designed for the methylation regions of the four gene promoters of MAL / SDC2 / TFPI2 / Vimentin, and the primer probe positions were designed for the methylation hotspot regions described in Example 1, and the designed primer probe sequence SEQ ID NO. 1-131 as shown in Table 6:

[0140] Table 6 Primer probe information

[0141]

[0142]

[0143]

[0144]

[0145] Use the nucleic acid extraction kit (product of Xiamen Aide Biomedical Technology Co., Ltd., product number: ADx-TI01c1814560) to extract DNA from the colorectal cancer cell line HCT15 / HCT116. The DNA extraction steps are as follows:

[0146] 1. Take 20-60mg of cell line samples and add 1mL of normal saline, shake and mix well, centrifuge at 1200g for 2min, carefully remove the supernatant;

[0147] 2. Add 180uL Buffer DTL, add 20uL Proteinase K Solution, vortex and mix, digest at 56°C until a relatively clear solution is obtained, which can be digested overnight;

[0148] 3. Take ou...

Embodiment 3

[0189] Compared directly using the QIAamp Fast DNA Stool Mini Kit (QIAGEN product, article number: 51604) to extract the stool sample DNA, then using the EpiTect Fast DNA Bisulfite Kit (QIAGEN company product, article number: 59826) to carry out bisulfite conversion, and the present invention in During the extraction process, two sample processing methods are completed: extraction, bisulfite conversion, washing purification and recovery. Quantus was used to detect the sample concentration, and the PCR reaction system was used to detect the sample recovery after different sample extraction and bisulfite conversion. Compare the advantages and disadvantages of each, and the specific operation steps are as follows.

[0190] Extract stool DNA first and then perform bisulfite conversion method: first use QIAamp Fast DNA StoolMini Kit (QIAGEN product, product number: 51604) to extract DNA from stool samples, and the extraction operation is as follows:

[0191] 1. Take 1g of feces sa...

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Abstract

The invention discloses a fecal detection kit for advanced adenoid tumor and colorectal cancer related gene multi-loci methylation detection. The fecal detection kit comprises a primer and a probe asfollows: SEQ ID NO: 1-SEQ ID NO:131 and further comprises a composition for greatly extracting and converting fecal DNA. The kit disclosed by the invention can be used for noninvasive fecal detectionof an intestinal cancer methylation related gene and early screening of patients suffering from advanced adenoid tumor and colorectal cancer of large intestines in a way such as multi-gene joint detection. Due to the adoption of multi-combination joint detection of the kit disclosed by the invention, a result is precise; the kit has the advanced adenoid tumor sensitivity of 62-90%, the colorectalcancer sensitivity of 78-100% and the specificity of 82-100% due to the adoption of single-combination single-tube detection, single-combination repeated-tube detection and multi-combination joint methylation detection; in addition, the total detection cost is lower than the sigmoidoscopy expense, and the kit can be used for various purpose such as early screening of colorectal cancer before clinic traditional detection and is a detection means capable of avoiding parts of subjects from suffering from pain caused by a sigmoidoscope and the like.

Description

technical field [0001] The invention belongs to the field of medical technology, and relates to a methylation multi-site combined detection kit for detecting advanced adenoma of the large intestine and early screening of colorectal cancer. Background technique [0002] In China, the incidence and mortality of colorectal cancer rank fifth among malignant tumors. The incidence of colorectal cancer is on the rise. In China, there are more than 400,000 new patients with colorectal cancer each year, and about 200,000 people die each year. For colorectal cancer, the domestic annual incidence rate is 4.2%, which is higher than the international level of 2%. Early detection and early treatment of colorectal cancer play a vital role in the prognosis, recurrence, and survival of patients. The 5-year OS of stage Ⅰ-ⅡA colorectal cancer is above 90%, and as the cancer progresses, the 5-year OS gradually decreases. The 5-year OS of colorectal cancer treatment is only 13%. The early symp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/154
Inventor 李有彩林喜建宋庆涛郑立谋吕唯张慧英秦秋平廖乃淞
Owner 杭州艾维克生物科技有限公司
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