Detection kit and detection method for methylation of septin9 gene in human peripheral blood cell-free DNA

A detection kit and gene detection technology, which is applied in the fields of biology and medicine, can solve the problems of sensitivity and invasiveness, and achieve the effect of improving the cure rate, facilitating clinical application, and reducing mortality

Inactive Publication Date: 2016-08-17
杭州壹锋生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the sensitivity or invasiveness of the above methods, there is a need for a more a

Method used

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  • Detection kit and detection method for methylation of septin9 gene in human peripheral blood cell-free DNA
  • Detection kit and detection method for methylation of septin9 gene in human peripheral blood cell-free DNA
  • Detection kit and detection method for methylation of septin9 gene in human peripheral blood cell-free DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1. Plasma separation and free DNA extraction

[0060] 1. Plasma Separation

[0061] 1) Collect 5mL-10mL peripheral blood with EDTA anticoagulant tube;

[0062] 2) Centrifuge the blood sample at 1,600g for 10 minutes;

[0063] 3) Carefully transfer the collected plasma sample to a 2mL centrifuge tube;

[0064] 4) Centrifuge the plasma sample at 4°C at 16,000g for 10 minutes;

[0065] 5) Carefully transfer the collected plasma sample to a new 2mL centrifuge tube;

[0066] 6) The collected plasma samples are stored at -20°C.

[0067] 2. Extraction of cell-free DNA

[0068] 1) Add 2mL Lysis Buffer, 100uL PK, 15uL sedimentation aid, 30uL magnetic beads (invert or use a pipette to mix the magnetic beads before use), 600-1300uL (preferably 1000uL) into a 5mL nuclease-free tube Sample, vortex to mix. At room temperature, mix up and down for 20 minutes to fully combine the magnetic beads and nucleic acid.

[0069] 2) Transfer the mixture to a 1.5mL centrifuge tub...

Embodiment 2

[0076] Example 2. Enzymatic hydrolysis of free DNA

[0077] 1) Add 30ng of free DNA, 1U of BstUI enzyme and buffer into a 0.5mL thin-walled tube, the total volume is 50uL.

[0078] 2) Water bath at 60°C for 10 hours.

[0079] 3) After enzymatic hydrolysis, methylated DNA will be retained, and unmethylated DNA will be enzymatically hydrolyzed.

Embodiment 3

[0080] Example 3. Detection of target gene by fluorescent quantitative PCR

[0081] Fluorescence quantitative PCR detection based on Taqman probe:

[0082] According to the following fluorescent quantitative PCR system, add the reagents to the 200 μL EP tube in the following order:

[0083]

[0084] After mixing, centrifuge slightly, and perform fluorescence quantitative PCR reaction in ABI 7500. The reaction parameters are set as:

[0085]

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Abstract

The invention belongs to the field of biomedicine, and provides a detection kit and a detection method for methylation of septin9 gene in human peripheral blood cell-free DNA. The kit comprises a primer sequence corresponding to a septin9 gene methylation detection site, a septin9 gene detection site probe sequence, a primer sequence corresponding to a reference gene detection site, and a reference gene detection site probe sequence, wherein the septin9 gene detection site is one site of septin9 gene CpG island 3, and the reference gene is Actin. The design and result reading of the detection kit provided by the invention are simple and convenient in the whole detection flow, and the detection result is reliable and high in specificity and sensitivity. By carrying out enzymolysis of cell-free DNA for detection of methylation of the septin9 gene, the sample loss is small. The method is simple, quick, economical and practical. The detection kit and detection method can be used for assisting in screening and diagnosis of carcinoma of large intestine, and further improving the cure rate and lowering death rate.

Description

technical field [0001] The invention belongs to the fields of biology and medicine, and in particular relates to a detection kit and a detection method for septin9 gene methylation in human peripheral blood free DNA. Background technique [0002] Colorectal cancer is a serious disease that occurs in the lower gastrointestinal system and is one of the three major malignant tumors in the world. There are 1.361 million new cases of colorectal cancer every year in the world, directly causing 694,000 deaths. With the improvement of people's living standards and changes in eating habits, the incidence of colorectal cancer is increasing, especially in large and medium-sized cities such as Beijing and Shanghai, and colorectal cancer has gradually become the most common malignant tumor besides lung cancer. The early symptoms of colorectal cancer are not obvious. Due to the weak awareness of cancer screening among the public and the rejection of existing detection techniques, most pa...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/154C12Q2600/166
Inventor 朱聪张启坤丁先锋
Owner 杭州壹锋生物科技有限公司
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