Kit for detecting methylation of colorectal cancer related gene multi-sites and application of kit

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: 2017-06-27
WUHAN AIMISEN LIFE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Kit for detecting methylation of colorectal cancer related gene multi-sites and application of kit
  • Kit for detecting methylation of colorectal cancer related gene multi-sites and application of kit
  • Kit for detecting methylation of colorectal cancer related gene multi-sites and application of kit

Examples

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Embodiment 1

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

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

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

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

Embodiment 2

[0079] This embodiment provides a kit and application for detecting multi-site methylation of colorectal cancer-related genes. The primer combination includes a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, and a fifth primer pair. primer pair, 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 pair 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. 9-10; the probe includes a first probe, a second probe, a third probe, a fourth probe and a fifth probe, and the base sequence of the first probe is as shown in SEQ ID As 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 probe is sho...

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 kit for methylation of colorectal cancer related gene multi-sites and application of the kit. A primer combination and a probe can specifically detect the methylation degrees of a plurality of colorectal cancer related gene sites. The invention further provides application and a method of the primer combination and the probe for detecting the methylation in colorectal cancer and precancerous polyps screening. The method comprises the steps that a cell lysis solution is added to cell sediment and mixed uniformly, then PVPP is added for crude extraction, a cell sediment sample after primary treatment contains a little protein and RNA, protease K and RNA enzyme are added for completely removing the protein and the RNA, cell lysate is added to a centrifugal column with an adsorption film, after impurities are washed by a DNA rising solution, a DNA eluent is added to elute the DNA in the absorption film, and finally a DNA solution is collected through centrifugation. Efficient pipe collection detection is performed aiming at the plurality of gene sites, the flexibility and the specificity are improved, and the tumor early screening requirement can be improved.

Description

technical field [0001] The invention relates to the field of medical technology, in particular to a kit and application for detecting multi-site methylation of colorectal cancer-related genes. Background technique [0002] Colorectal cancer is the third most common malignant tumor in the world. In recent years, the incidence of colorectal cancer in China has been increasing year by year, and 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-stage colorectal cancer can be as high as 90%, and the 5-year survival rate of patients with intermediate and advanced stage is only about 10%. One of the key factors to keep high. The morbidity and mortality of early-stage colorectal cancer patients who have been effectively treated are reduced by 60% and 80%. Therefore, early detection, early diagnosis and early treatment of colorectal cancer are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
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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