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HER2 (human epidermal growth factor receptor-2) detection kit and detection method and application thereof

A technology of kits and probes, applied in the fields of medicine and biology, to achieve the effects of avoiding delays in patients' conditions, high accuracy, and ensuring accuracy

Pending Publication Date: 2019-08-16
深圳海普洛斯医学检验实验室
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the accuracy of the detection method needs to be further improved

Method used

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  • HER2 (human epidermal growth factor receptor-2) detection kit and detection method and application thereof
  • HER2 (human epidermal growth factor receptor-2) detection kit and detection method and application thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0077] Example 1 Extraction of paraffin-embedded pathological section tissue DNA

[0078] In this example, a nucleic acid extraction and purification kit (QIAamp DNA FFPE Tissue Kit 56404) was used for DNA extraction, and the specific steps are as follows:

[0079] (1) Prepare buffers AW1 and AW2, check whether absolute ethanol is added to the reagents, mix well, and set aside;

[0080] (2) Turn on the metal bath, adjust the temperature to 56°C, and set it aside;

[0081] (3) Take the corresponding number of 2mL centrifuge tubes and number them;

[0082] (4) Paraffin tissue section (FFPE) samples were scraped with a scalpel blade, among which, 5 pieces of each paraffin tumor tissue section sample were scraped and placed in corresponding numbered 2mL centrifuge tubes, and 10 pieces of each paraffin puncture tissue sample were scraped and placed in corresponding In a numbered 2mL centrifuge tube;

[0083] (5) Add 160 μL of deparaffinization solution (Deparaffinization Solutio...

Embodiment 2

[0098] Example 2 Extraction of peripheral blood cfDNA

[0099]The cfDNA content in peripheral blood is very low, the ratio is about 1 / 1000, so it is very important to ensure the extraction quality of plasma cfDNA. In this example, a nucleic acid extraction and purification kit (ZD-YL-Midi-40) was used for cfDNA extraction, and the specific steps are as follows:

[0100] (1) Add 20 μL of proteinase K to a 10 mL centrifuge tube, then add 2 mL of separated plasma, and 2 mL of buffer GH, vortex and mix for 15 s, and place the mixture in a constant temperature water bath at 37 ° C for 10 min;

[0101] (2) Add 2 mL of isopropanol (plasma: GH: isopropanol = 1:1:1) to the above mixture, vortex fully and incubate in a refrigerator at 4°C for 10 min, then transfer the mixture into a collection tube Centrifuge at 10,000rpm for 1min after the medium-volume spin column, and discard the waste liquid in the collection tube;

[0102] (3) Add 2.5mL buffer W1A (add absolute ethanol as require...

Embodiment 3

[0112] Example 3 HER2 gene amplification detection

[0113] In this embodiment, the primer-probe composition as shown in SEQ ID NO: 1-18 is used to detect the amplification mutation of HER2 gene, wherein the primers of HER2 gene are SEQ ID NO: 1-6, and the probe is SEQ ID NO: 7 ~9, the primers of the internal reference gene CEP17 are SEQ ID NO:10-13, the probe is SEQ ID NO:14-15, the primers of the internal reference gene GAPDH are SEQ ID NO:16-17, and the probe is SEQ ID NO:18. The primers and probes used in this example were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0114] Exemplarily, this embodiment uses the peripheral blood cfDNA extracted in Example 2 for detection, and the specific steps are:

[0115] (1) Configure the reaction system:

[0116] Configure the PCR reaction system according to Table 1 (without adding cfDNA template first), and aliquot 12 μL / tube. This stage is completed in the reagent preparation area, and the prepared reaction system i...

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Abstract

The invention provides an HER2 (human epidermal growth factor receptor-2) detection kit and a detection method and application thereof. Primers of HER2 are shown in SEQ ID NO.:1-6; probes of the HER2are shown in SEQ ID NO.:7-9. The primers and the probes are designed for HER2 genes, which cooperates with primers and probes of internal reference genes CEP17 and GAPDH, the amplification mutation ofDNA genes in paraffin embedded pathological section or cfDNA genes in peripheral plasma of patients with breast cancer is detected quantitatively according to the ratio of HER2 genes to the internalreference genes, and key information is provided for targeted drug use; the HER2 detection kit and the detection method and application thereof have the advantages of absolute quantification, short detection cycle, high specificity, high accuracy, high sensitivity and the like.

Description

technical field [0001] The invention belongs to the field of medicine and biotechnology, and relates to a kit and its detection method and application, in particular to a HER2 detection kit and its detection method and application. Background technique [0002] Breast cancer is a malignant tumor that seriously threatens women's health. It has the highest morbidity and mortality among female patients. In recent years, the number of breast cancer patients in my country has continued to increase at a rate of 3% to 7%. Accurate diagnosis and individualized treatment can significantly prolong patient survival, improve patient quality of life and reduce mortality. [0003] Human genomics studies have found that the amplification of human epidermal growth factor receptor 2 (human epidermal growth factor receptor-2, HER2) can promote the expression of HER2 protein on the surface of tumor cells, leading to the overactivity of HER2-dominated growth signals, and ultimately triggering c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6886C12Q2600/156C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/101C12Q2563/159
Inventor 张晓妮李慧刘园园许明炎
Owner 深圳海普洛斯医学检验实验室
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