Reagent kit for identifying breast cancer states or breast precancerous lesion and application of reagent kit

A breast cancer and reagent kit technology, applied in the biological field, achieves the effects of rapid diagnosis and evaluation, improved sensitivity and specificity, and guaranteed accuracy and reliability

Inactive Publication Date: 2018-12-25
BEIJING EXELLON MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of means to effectively detect the methylat

Method used

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  • Reagent kit for identifying breast cancer states or breast precancerous lesion and application of reagent kit
  • Reagent kit for identifying breast cancer states or breast precancerous lesion and application of reagent kit
  • Reagent kit for identifying breast cancer states or breast precancerous lesion and application of reagent kit

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0059] Example 1: DNA extraction

[0060] DNA extraction reagents consist of lysis buffer, binding buffer, washing buffer and elution buffer. The lysis buffer is composed of protein denaturant, detergent, pH buffer and nuclease inhibitor. The binding buffer consists of a protein denaturant and a pH buffer. The cleaning buffer is divided into cleaning buffer A and cleaning buffer B. Cleaning buffer A is composed of protein denaturant, nuclease inhibitor, detergent, pH buffer and ethanol; cleaning buffer B is composed of nuclease inhibitor, Composition of pH buffer and ethanol. The elution buffer consists of a nuclease inhibitor and a pH buffer. The protein denaturant is: guanidine hydrochloride; the detergent is: Tween20; the pH buffer is: Tris-HCl; the nuclease inhibitor is: EDTA.

[0061] In this embodiment, a plasma sample of a breast cancer patient is taken as an example to extract plasma DNA. The extraction method includes the following steps:

[0062] (1) Take 1ml of plasm...

Example Embodiment

[0074] Example 2: Bisulfite treatment of DNA

[0075] Bisulfite treatment of DNA is processed with a bisulfite reagent. The bisulfite reagent is composed of a bisulfite buffer and a protection buffer; the bisulfite buffer is a mixed liquid of sodium bisulfite and water ; The protection buffer is a mixed liquid of the oxygen free radical scavenger hydroquinone and water.

[0076] In this example, the DNA extracted in Example 1 is used as the processing object, and the DNA is processed with bisulfite. The specific steps include:

[0077] (1) Preparation of bisulfite buffer solution: Weigh 1g of sodium bisulfite powder and add water to prepare a 3M buffer solution;

[0078] (2) Prepare protection buffer: Weigh 1g hydroquinone reagent, add water to prepare 0.5M protection buffer;

[0079] (3) Mix 100μl DNA solution, 200μl bisulfite buffer solution and 50μl protection solution, shake and mix well;

[0080] (4) Thermal cycle: 95°C for 5 minutes, 80°C for 60 minutes, 4°C for 10 minutes;

[0081...

Example Embodiment

[0091] Example 3: Real-time fluorescent PCR detection of DNA methylation and primer set verification

[0092] In this embodiment, real-time fluorescent PCR is used as an example to measure the methylation level of biomarker genes. The detected genes are APC, B RCA1, CCND2, CST6, GP5, GSTP1, PITX2, RARB, RASSF1A and SOX17 genes, and the internal reference gene is ACTB. This example uses the bisulfite-treated DNA of Example 2 as a template for real-time fluorescent PCR amplification. The DNA sample to be tested, the negative control product, the positive quality control product and the no-template control are all tested in 3 replicates.

[0093] For APC, BRCA1, CCND2, CST6, GP5, GSTP1, PITX2, RARB, RASSF1A and S OX17 genes, many sets of primers and probe combinations can be designed, and the performance of each set of probe primer combinations may be different, so Verify through experiments.

[0094] Therefore, the present invention has designed a variety of primers and probes for A...

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Abstract

The invention relates to a reagent kit for identifying breast cancer states or breast precancerous lesion and application of the reagent kit. The reagent kit comprises primer pairs for detecting methylation levels of biological marker genes or corresponding segments in biological samples from subjects. The primer pairs are used for carrying out PCR (polymerase chain reaction) by the aid of the biological marker genes or the segments of the biological marker genes, the biological marker genes or the segments are used as templates, and the biological marker genes are treated by hydrosulfite; thebiological marker genes are selectively a type or a plurality of types of APC, BRCA1, CCND2, CST6, GP5, GSTP1, PITX2, RARB, RASSF1A and SOX17. The reagent kit and the application have the advantagesthat the methylation levels of the biological marker genes or the corresponding segments are jointly detected, the sensitivity and the specificity of breast precancerous lesion and breast cancer detection can be improved, accordingly, the correctness and the reliability of detection results can be guaranteed, and a novel quick, reliable and accurate way can be provided to predicting, diagnosing and evaluating breast cancer.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for identifying breast cancer status or pre-cancerous lesion and application thereof. Background technique [0002] Breast cancer is the most common malignant tumor in women. The probability of breast cancer in a woman's life is 10%. There are about 1.3 million breast cancer patients in the world every year, and about 400,000 people die from the disease, and the annual rate is 2%~ 3% speed increments. In the United States, the incidence of breast cancer ranks first in female malignant tumors, and its mortality rate ranks second in the mortality rate of malignant tumors. In my country, the incidence of breast cancer has also leapt to the first place among female malignant tumors, and the age of onset is becoming younger. Breast cancer has become the number one killer of women worldwide. The survival period of breast cancer patients is closely related to the pathological stage...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/154
Inventor 徐春叶蒲珏李明明陈彦利李淑钰
Owner BEIJING EXELLON MEDICAL TECH CO LTD
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