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Reagent kit for detecting Septin9 gene methylation and application of reagent kit

A kit and methylation technology are applied in the field of kits for detecting the methylation of Septin9 gene in colorectal cancer, which can solve the problems of low penetration rate, difficulty in clinical promotion and popularization, inconvenience in the promotion of detection methods, etc., and achieve high extraction efficiency. , The effect of reducing the cost of artificial reagents and reducing the amount of plasma

Pending Publication Date: 2016-11-23
BIOCHAIN BEIJING SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Epigenomics AG’s Septin9 gene methylation detection kit Epi proColon 2.0CE provides a method for detecting Septin9 gene methylation by PCR fluorescent probe method, but this method has many inconveniences in clinical detection. If the amount of plasma is used in a large amount, the patient has resistance, which is not convenient for the promotion of the detection method; the applicable models are ABI 7500fast / fast DX and Roche 480I / II, and the penetration rate of the above-mentioned models is very low, which has caused many hospitals to fail It is more difficult to carry out the test, clinical promotion and popularization

Method used

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  • Reagent kit for detecting Septin9 gene methylation and application of reagent kit
  • Reagent kit for detecting Septin9 gene methylation and application of reagent kit
  • Reagent kit for detecting Septin9 gene methylation and application of reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 : kit composition

[0057] The materials required for the detection experiment are shown in Table 1 and Table 2.

[0058] 1. Kit composition:

[0059] Table 1 Kit I: Plasma Processing Kit

[0060]

[0061] Table 2 Kit II: colorectal cancer methylation gene detection kit

[0062]

[0063] illustrate:

[0064] 1) In the kit I, except that the lysis adsorption solution, the concentrated washing solution A and the magnetic beads are the preferred reagents replaced by the present invention, other reagents are purchased from Epigenomics Epi ProColon; in the kit II, the Taq enzyme is the preferred replacement of the present invention Reagents, others were purchased from Epigenomics Epi ProColon.

[0065] 2) Magnetic beads T were purchased from BioChain Institute, Inc.

[0066] 3) The lysis adsorption solution and the concentrated lotion A are self-prepared. The formula of the lysis adsorption solution is shown in Table 3, and the concentrated lotion A is...

Embodiment 2

[0074] Example 2 : Extraction of plasma cell-free DNA

[0075] Compared with the extraction method of Epi ProColon 3.5mL plasma volume, the present invention reduces the plasma volume to 1.5-1.75mL. On the one hand, it reduces the patient's resistance during clinical blood drawing, and at the same time, the present invention improves the DNA extraction method technically. , effectively improving the recovery rate of DNA, which is more conducive to PCR detection. Specific improvements include:

[0076] 1) The establishment of the optimal reagent dosage for each step. By testing negative and positive reference substances and plasma contrast tests, the most suitable detection method for 1.5-1.75mL plasma volume was established, which significantly reduced the reagent cost for testing the same sample.

[0077] 2) Through the introduction of step 39), the purity of the recovered DNA solution is effectively improved, and the efficiency of the PCR reaction and the sensitivity of d...

Embodiment 3

[0123] Example 3 : Detection of methylated Septin9 gene

[0124] Compared with Epi ProColon, the invention reduces the amount of plasma samples, reduces the complexity and cost of detection by expanding the detection volume and optimizing the reaction system, and improves detection efficiency, cost performance and compliance. Detection method of the present invention is as follows:

[0125] 1) Prepare PCR pre-reaction solution according to Table 5

[0126] Table 5 Preparation of PCR pre-reaction solution

[0127]

[0128] 2) Shake and mix the PCR pre-reaction solution, add 30 μl to the selected 96-well plate.

[0129] 3) Add 30 μl of sulfite-converted DNA (BisDNA) to the corresponding well of the PCR plate, and use Optical Adhesive Film film seal.

[0130] 4) Centrifuge at 1000 rcf for 1 minute.

[0131] 5) Run the ABI7500 fluorescent PCR instrument, and open the 7500 Software V2.0.5 software.

[0132] 6) Click "New Document" → click the "Experiment Properties" tab...

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Abstract

The invention relates to a reagent kit for detecting Septin9 gene methylation. The reagent kit comprises PCR reaction liquid and polymerase. The PCR reaction liquid is composed of dNTP, primers, probes and a solvent. The polymerase is Taq polymerase BCIUS2. The invention further provides a method for extracting plasma DNA with the reagent kit and application of the reagent kit in detecting Septin9 gene methylation. By means of the detection method and the reagent kit, colorectal cancer Septin9 gene methylation can be easily, conveniently and rapidly detected; the detection method and the reagent kit are high in specificity and sensitivity and can be applied to clinical auxiliary diagnosis of colorectal cancer and clinical early screening of colorectal cancer patients.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a kit for detecting the methylation of the Septin9 gene in colorectal cancer. Background technique [0002] Colorectal cancer is a common malignant tumor, which affects about 900,000 people every year worldwide, and is one of the most common malignant tumors of the digestive tract. Colorectal malignant tumors originate from mucosal epithelium or submucosal mesenchymal tissue. Among them, malignant tumors arising from the epithelium of the large intestine mucosa under the action of various carcinogenic factors such as environment or genetics are collectively called colorectal cancer. Colorectal cancer occurs in the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum. It is a general term for colon cancer and rectal cancer, also known as colorectal cancer. [0003] Colorectal cancer is a malignant tumor that seriously threatens human life and...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/10
Inventor 韩晓亮周光朋吴振张晓姣王建铭
Owner BIOCHAIN BEIJING SCI & TECH
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