Multi-site methylation kit for detecting colorectal cancer-related genes and its application

A colorectal cancer and kit technology, applied in the medical field, can solve problems such as inability to detect multiple gene loci, and achieve the effect of facilitating subsequent detection and improving sensitivity and specificity

Active Publication Date: 2021-04-30
WUHAN AIMISEN LIFE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 2. This method has limitations on the capture of DNA fragments of the target gene. It can only capture the sequence of specific fragments of magnetic beads, and cannot detect multiple gene loci.

Method used

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  • Multi-site methylation kit for detecting colorectal cancer-related genes and its application
  • Multi-site methylation kit for detecting colorectal cancer-related genes and its application
  • Multi-site methylation kit for detecting colorectal cancer-related genes and its application

Examples

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Embodiment 1

[0058] The present invention proposes a multi-site methylation kit for detecting colorectal cancer-related genes and its application. The fecal genome extraction kit is used for DNA separation and extraction to obtain the fecal genome. The steps are as follows:

[0059] 1. Use a special feces collection box to collect the feces of hospital patients, which contains a fecal genome preservation solution, and the preservation solution contains 0.1mol / L EDTA and 0.5mol / L TRIS, and its function is to protect the genomic DNA from the nucleic acid in the feces enzymatic degradation.

[0060] 2. In this example, the feces sample was fully shaken and mixed in the sample preservation solution, and then 1.5ml of the mixed solution was taken into a 2.0ml EP tube, and 1 / 9 of the 40% SDS lysate of the sample was added, and 8 μL of proteinase K was added. After shaking and mixing, lyse at 70°C for 10 minutes.

[0061] 3. Centrifuge at 12000rpm for 8min, transfer 900μL supernatant to a new EP...

Embodiment 2

[0079] This embodiment provides a multi-site methylation kit and application for detecting colorectal cancer-related genes. The primer combination includes the first primer pair, the second primer pair, the third primer pair, the fourth primer pair and the fifth primer pair. A pair of primers, the base sequence of the first primer pair is shown in SEQ ID NO.1-2, the base sequence of the second primer pair is shown in SEQ ID NO.3-4, the third primer The base sequence of the pair is shown in SEQ ID NO.5-6, the base sequence of the fourth primer pair is shown in SEQ ID NO.7-8, and the base sequence of the fifth primer pair is shown in SEQ ID NO Shown in .9-10; Described probe comprises first probe, second probe, the 3rd probe, the 4th probe and the 5th probe, the base sequence of described first probe is as SEQ ID Shown in NO.11, the sequence of the second probe is shown in SEQ ID NO.12, the sequence of the third probe is shown in SEQ ID NO.13, and the sequence of the fourth prob...

Embodiment 3

[0111] The invention provides a multi-site methylation kit for detecting colorectal cancer-related genes and its application. The kit also includes a sample preservation solution, a nucleic acid extraction solution, a PCR reaction solution, a positive quality control product, a negative quality control product, One or more of internal standard controls and standard reagents.

[0112] The kit also includes one or more of a dissolution buffer, a methylation conversion reagent, a binding buffer, and a washing buffer, which can perform CT conversion on the above-mentioned purified DNA.

[0113] The dissolution buffer includes 1-5mol / L NaOH and 5-20mmol / L hydrogen, preferably 3mol / L NaOH and 10mmol / L hydrogen have the best effect.

[0114] The methylation conversion reagents include but are not limited to salts containing sulfite groups such as sodium bisulfite, sodium sulfite, ammonium bisulfite and ammonium sulfite, with a concentration of 1-10mol / L, preferably ammonium bisulfite...

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Abstract

The invention discloses a multi-site methylation kit for detecting colorectal cancer-related genes and its application. The primer combination and probe provided by the invention can specifically detect the methylation of multiple colorectal cancer-related gene sites. degree of transformation. The invention also provides the primer combination for detecting methylation, the application of the probe in the screening of colorectal cancer and precancerous polyps and the method thereof. It includes the following steps: add cell lysate to the cell pellet and mix well, then add PVPP for rough extraction, there is still a small amount of protein and RNA in the cell pellet sample after preliminary treatment, then add proteinase K and RNase to completely remove protein and RNA For RNA, the cell lysate is added to the spin column with the adsorption membrane, after the impurities are washed by the DNA washing solution, the DNA eluent is added to elute the DNA from the adsorption membrane, and finally the DNA solution is collected by centrifugation. Efficient combined detection of multiple gene loci improves the sensitivity and specificity, and can meet the requirements of early tumor screening.

Description

technical field [0001] The invention relates to the field of medical technology, in particular to a kit for detecting multi-site methylation of colorectal cancer-related genes and its application. Background technique [0002] Colorectal cancer is the third most common malignancy in the world. In recent years, the incidence of colorectal cancer in China has been increasing year by year. It is estimated that there are about 400,000 new cases each year, ranking second among malignant tumors of the digestive system in my country. The 5-year survival rate of patients with early colorectal cancer can be as high as 90%, while the 5-year survival rate of patients with middle and advanced stages is only about 10%. However, about 80% of clinically diagnosed colorectal cancers are middle and late stages, which leads to the highest mortality rate. one of the key factors. The incidence rate of patients with early colorectal cancer after effective treatment is reduced by 60%, and the m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6806C12Q1/6858C12N15/11
CPCC12Q1/6806C12Q1/6858C12Q1/6886C12Q2600/154C12Q2523/308C12Q2521/537C12Q2521/327C12Q2531/113C12Q2535/122C12Q2563/107
Inventor 张良禄董兰兰姚希辉
Owner WUHAN AIMISEN LIFE TECH CO LTD
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