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A kit and method for detecting her-2 gene copy number variation based on digital PCR technology

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

Active Publication Date: 2019-07-12
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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  • A kit and method for detecting her-2 gene copy number variation based on digital PCR technology
  • A kit and method for detecting her-2 gene copy number variation based on digital PCR technology
  • A kit and method for detecting her-2 gene copy number variation based on digital PCR technology

Examples

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Effect test

Embodiment 1

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

[0032] 1. Sequence acquisition: the HER-2, CEP17, EFTUD2, EIF2C1 gene detection sites involved in the present invention are shown in Table 1.

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

[0034] gene Gene locus region HER-217q12 CEP1717p11.2 EFTUD217q21.31 EIF2C11p34.3

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

[0036] Table 2. Sequence list of HER-2, CEP17, EIF2C1, EFTUD2 primers and detection probes

[0037] Gene locus Primer and probe name serial number Sequence (5’-3’) HER-2-1F Forward primer 1 SEQ ID No. 1 GGTAGAACCTTTGCTGTC HER-2-1R Reverse primer 1 SEQ ID No. 2 GCAGAACCATACTGATGA HER-2-1P Detection probe 1 SEQ ID No. 3 TGTTCACCACTCTACCTCCAGC HER-2-2F Forward primer 2 SEQ ID No. 4...

Embodiment 2

[0039] Embodiment 2: A kit and method for detecting HER-2 gene copy number variation based on digital PCR technology is used to detect the HER-2 gene copy number variation in a paraffin tissue section sample.

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

[0041] 2. Method: Use Naica Crystal Digital TM Digital PCR instrument (STILLA Technologies, France) for digital PCR detection.

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

[0043] (2) Prepare the digital PCR reaction solution according to Table 3. After the preparation is completed, the PCR reaction tube i...

Embodiment 3

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

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

[0065] 2. Method: Use Naica Crystal Digital TM Digital PCR machine (STILLA Technologies, France) for testing.

[0066] (1) Sample collection: Use venipuncture for blood samples, and collect 10 mL of blood samples with STRECK BCT tube or EDTA anticoagulation tube (purple); if plasma separation cannot be performed within 2 hours, cell-free DNA protection is required when whole blood is collected The special room temperature blood collection tube (such as Streck BCT tube) for anti-cell lysis protective agent.

[0067] (2) Storage and transportation of blood samples: STRECK BCT tubes are blood collection tubes used for transportation and short-term storage within the specified temperature range (...

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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 a method for detecting the copy number variation of HER-2 gene based on digital PCR technology, and belongs to the technical field of biomedical nucleic acid detection. Background technique [0002] Breast cancer is the number one common malignant tumor among 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 mortality rate has exceeded 70,000. According to the results of many years of research, about 20% of breast cancer patients have increased levels of human epidermal growth factor receptor-2 (HER-2) gene or protein. Based on the results of patient follow-up investigations, the clinical application of trastuzumab (Herceptin), a breast cancer targeted drug targeting HER-2 gene amplification, can significantly improve the prognosis of patients with HER-2 positive breast cancer, thereby Changing the traditional treatment...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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