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Kit for detecting copy number variation of HER-2 gene by virtue of digital PCR technique and method

A HER-2, gene copy number technology, applied in the field of biomedical nucleic acid detection, can solve problems such as low accuracy

Active Publication Date: 2018-09-07
北京爱普拜生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to provide a kit and method for detecting HER-2 gene copy number variation based on digital PCR technology, so as to solve the technical problem of low accuracy of HER-2 gene copy number variation determination method in the prior art

Method used

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  • Kit for detecting copy number variation of HER-2 gene by virtue of digital PCR technique and method
  • Kit for detecting copy number variation of HER-2 gene by virtue of digital PCR technique and method
  • Kit for detecting copy number variation of HER-2 gene by virtue of digital PCR technique and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Design of specific PCR primers and detection probe sequences for detecting HER-2 gene copy number variation

[0032] 1. Sequence acquisition: See Table 1 for the detection sites of HER-2, CEP17, EFTUD2, and EIF2C1 genes involved in the present invention.

[0033] Table 1, HER-2, CEP17, EFTUD2, EIF2C1 gene detection sites

[0034] Gene

[0035] 2. Design method: According to the HER-2, CEP17, EIF2C1, and EFTUD2 gene sequences published in the NCBI database, specific primers and detection probes were designed (Table 2).

[0036] Table 2, HER-2, CEP17, EIF2C1, EFTUD2 primer and detection probe sequence list

[0037] Gene loci

[0038] 3. Design primers and detection probes based on the specific sequences obtained above. The primers and detection probes used in the patent of the present invention were synthesized at Thermo Fisher Scientific Corporation (Shanghai), USA.

Embodiment 2

[0039] Example 2: Using a kit and method for detecting HER-2 gene copy number variation based on digital PCR technology to detect HER-2 gene copy number variation in paraffin tissue section samples.

[0040] 1. Materials: Paraffin section samples of 18 breast cancer patients who have been identified by IHC or FISH.

[0041] 2. Method: use Naica Crystal Digital TM Digital PCR instrument (French STILLA Technologies company) carries out digital PCR detection.

[0042] (1) Nucleic acid extraction: It is recommended to use paraffin section DNA extraction kit (QIAGEN, USA, CatNo. 56404) for DNA extraction. The extraction step was carried out according to the product instructions, and 50 μL of DNA solution was collected and directly tested or stored at -20°C. At the same time, negative and positive quality control products were tested.

[0043] (2) The digital PCR reaction solution was prepared according to Table 3. After the preparation was completed, the PCR reaction tube was vo...

Embodiment 3

[0063] Example 3: Using a kit and method for detecting HER-2 gene copy number variation based on digital PCR technology to detect HER-2 gene copy number variation in blood samples.

[0064] 1. Materials: Randomly select blood samples from 5 of the 18 breast cancer patients in Example 2

[0065] 2. Method: use Naica Crystal Digital TM A digital PCR instrument (French STILLA Technologies company) was used for detection.

[0066] (1) Sample collection: blood samples are collected by venipuncture, and 10 mL of blood samples are collected using STRECK BCT tubes or EDTA anticoagulant tubes (purple); if plasma separation cannot be performed within 2 hours, a protective tube containing free DNA is required when collecting whole blood. Special room temperature blood collection tubes (such as Streck BCT tubes) with agents and anti-cell lysis protection agents.

[0067] (2) Preservation and transportation of blood samples: STRECK BCT tubes belong to the blood collection tubes used for ...

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Abstract

The invention provides a kit for detecting the copy number variation of an HER-2 gene by virtue of a digital PCR technique and a method. The kit comprises two groups of primer pairs and detection probes, a primer pair, a detection probe, a digital PCR premixed solution, luciferase, ddH2O and a calibration file, wherein the primer pairs and the detection probes are used for detecting the HER-2 gene, and the primer pair and the detection probe are used for detecting three reference genes CEP17, EIF2C1 and EFTUD2. Besides, the invention further provides a method for detecting the copy number variation of the HER-2 gene by virtue of the digital PCR technique. According to the method, phenomena of 17# chromosome multimer, aneuploid and centromere amplification can be eliminated, and the methodcan be used for accurately detecting whether the HER-2 gene is amplified to be positive or negative.

Description

technical field [0001] The invention relates to a kit and method for detecting HER-2 gene copy number variation based on digital PCR technology, belonging to the technical field of biomedical nucleic acid detection. Background technique [0002] Breast cancer is the number one common malignant tumor in women. According to statistics from the National Cancer Center in 2016, the number of new breast cancer cases nationwide reached 272,400, and the annual death rate has exceeded 70,000. According to the results of years of research, about 20% of breast cancer patients have increased human epidermal growth factor receptor-2 (Human Epidermal growth factor Receptor-2, HER-2) gene or protein levels. Based on the results of patient follow-up surveys, the clinical application of HER-2 gene amplification breast cancer targeting drug trastuzumab (Herceptin) can significantly improve the prognosis of HER-2 positive breast cancer patients, thereby It has changed the traditional treatme...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/156C12Q2600/166
Inventor 龚建冯晓燕林挺于祥春郑祖亮
Owner 北京爱普拜生物技术有限公司
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