Kit for detecting human MLH1 gene methylation and use method of kit

A technology of methylation and kit, which is applied in the detection kit of human MLH1 gene methylation and its application field, and can solve the problems of low specificity and sensitivity of the detection method

Pending Publication Date: 2021-02-23
深圳市新合生物医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The purpose of the present invention is to provide a blood single gene detection primer probe combination kit for early diagnosis, dete

Method used

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  • Kit for detecting human MLH1 gene methylation and use method of kit
  • Kit for detecting human MLH1 gene methylation and use method of kit
  • Kit for detecting human MLH1 gene methylation and use method of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Using the kit of the present invention to detect colorectal cancer methylation qPCR on healthy human cfDNA samples

[0062] (1) Experimental materials

[0063] 1. 5 parts of healthy human plasma;

[0064] 2. QIAamp Circulatory System Nucleic Acid Extraction Kit (purchased from Qiagen);

[0065] 3. EZ DNA Methylation-Lightning Kit (purchased from Zymo Research);

[0066] 4. The kit of the present invention (which includes primer and probe combination 1, ACTB amplification primer and probe for detecting target MLH1);

[0067] 5. AmpliTaq Gold TM DNA polymerase and buffer reagents (purchased from Thermo Fisher).

[0068] (2) Experimental method

[0069] 1. DNA extraction from plasma

[0070] cfDNA was extracted from 5 healthy human plasma samples using the QIAamp Circulatory System Nucleic Acid Extraction Kit, and the steps were performed according to the kit instructions.

[0071] 2. Methylation conversion of free DNA

[0072] Five extracted plasma cfD...

Embodiment 2

[0083] Example 2: Using the kit of the present invention to detect colorectal cancer methylation qPCR on ctDNA samples from colorectal cancer patients (1) Experimental materials

[0084] 1. 5 parts of blood plasma from patients with colorectal cancer;

[0085] 2. QIAamp Circulatory System Nucleic Acid Extraction Kit (purchased from Qiagen);

[0086] 3. EZ DNA Methylation-Lightning Kit (purchased from Zymo Research);

[0087] 4. The kit of the present invention (which includes primer and probe combination 1, ACTB amplification primer and probe for detecting target MLH1);

[0088] 5. AmpliTaq Gold TM DNA polymerase and buffer reagents (purchased from Thermo Fisher).

[0089] (2) Experimental method

[0090] 1. DNA extraction from plasma

[0091] ctDNA was extracted from 5 plasma samples of patients with colorectal cancer using the QIAamp Circulatory System Nucleic Acid Extraction Kit, and the steps were performed according to the kit instructions.

[0092] 2. Methylation ...

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Abstract

The invention discloses a kit for detecting human MLH1 gene methylation and a use method of the kit. The kit comprises a specific primer and a probe which are used for detecting or measuring the methylation state or level of the MLH1 gene in DNA of a tested sample. The kit can significantly increase the positive detection rate and specificity (real negative rate) of early colorectal cancer and canimprove the detection sensitivity of a biomarker to a picogram/nanogram level DNA molecule, and the improvement of the detection sensitivity is realized by optimizing a specific nucleotide sequence primer and a DNA probe and improving a bisulfite treatment method of plasma free DNA.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and DNA detection, and in particular relates to a DNA methylation qPCR kit for colorectal cancer detection and its application method, in particular to a method for methylation qPCR using plasma free DNA A kit for detecting or screening colorectal cancer by determining the methylation status of one or more target gene targets (such as MLH1) and a method of use thereof. Background technique [0002] Colorectal cancer (CRC) is the third most common cancer worldwide, killing 600,000 people each year. For CRC, early recognition and early intervention are the best ways to reduce mortality. The current screening method is colonoscopy with fecal occult blood and immunohistochemical testing (gFOBT / FIT). These screening methods are underutilized due to the discomfort of colonoscopy and the distaste for bowel products. Therefore, assessment of CRC risk using reliable non-invasive blood-based tests ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2600/166C12Q2531/113C12Q2563/107C12Q2523/125C12Q2525/113C12Q2561/101
Inventor 王志强万季蔡雪儿罗海燕王一杏夏迪汪健金泰庆关建洪王弈宋麒
Owner 深圳市新合生物医疗科技有限公司
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