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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 methylation status of these cancer-related genes and process the detection results

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
Effect test

Embodiment 1

[0059] Example 1: DNA Extraction

[0060] DNA Extraction Reagent consists of Lysis Buffer, Binding Buffer, Washing Buffer and Elution Buffer. Lysis buffer consists of protein denaturants, detergents, pH buffers and nuclease inhibitors. Binding buffers consist of protein denaturants and pH buffers. The cleaning buffer is divided into cleaning buffer A and cleaning buffer B, and cleaning buffer A is composed of protein denaturant, nuclease inhibitor, detergent, pH buffer and ethanol; cleaning buffer B is composed of nuclease inhibitor, pH buffer and ethanol composition. The elution buffer consists of nuclease inhibitors and pH buffers. The protein denaturant is: guanidine hydrochloride; the detergent is: Tween20; the pH buffer is: Tris-HCl; the nuclease inhibitor is: EDTA.

[0061] This embodiment takes the plasma sample of a breast cancer patient as an example to extract plasma DNA. The extraction method comprises the following steps:

[0062] (1) Take 1ml of plasma, add ...

Embodiment 2

[0074] Example 2: Bisulfite Treatment of DNA

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

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

[0077] (1) Preparation of bisulfite buffer: weigh 1g of sodium bisulfite powder, add water to prepare 3M buffer;

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

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

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

[0081] (5) Add 1ml DNA bi...

Embodiment 3

[0091] Example 3: Detection of DNA methylation by real-time fluorescent PCR and verification of primer sets

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

[0093] For APC, BRCA1, CCND2, CST6, GP5, GSTP1, PITX2, RARB, RASSF1A and SOX17 genes, many sets of primer and probe combinations can be designed, and the performance of each set of probe primer combinations may be different, so it is necessary to Verify by experiment.

[0094] Therefore, the present invention designs a v...

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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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