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Detection primer combination and kit for HER2 (human epidermal growth factor receptor 2) gene amplification

A detection kit and gene amplification technology, applied in the field of genetic detection, can solve problems such as loss of individualized medication, inaccessibility of personnel, and expensive equipment, and achieve timely diagnosis and treatment of cases and monitoring of treatment effects, effective and accurate detection, and low cost low effect

Inactive Publication Date: 2015-12-02
北京鑫诺美迪基因检测技术有限公司
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, FISH has the limitations of expensive instruments, long detection cycle, high patient fees, professional knowledge of testers and laboratory conditions. The detection reagents have been monopolized by one or two domestic or foreign manufacturers, and can only be carried out in qualified large hospitals. , resulting in a large number of people not being able to get a timely diagnosis and losing the opportunity for individualized medication. Therefore, there is an urgent need for a detection method with advantages such as high sensitivity, accuracy, easy operation, standardization, and diversified detection.

Method used

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  • Detection primer combination and kit for HER2 (human epidermal growth factor receptor 2) gene amplification
  • Detection primer combination and kit for HER2 (human epidermal growth factor receptor 2) gene amplification

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Embodiment 1

[0052] 1. Design of primers and probes

[0053] Design specific primers HER2(R)-F and HER2(R)-R for the receptor domain of HER2 transcriptome, and Taqman fluorescent probe HER2(R)-FP. The fluorescent reporter group labeled at the 5' end of the Taqman fluorescent probe HER2(R)-FP is 6-carboxyfluorescein (6-carboxyfluorescein, FAM), and the quencher group labeled at the 3' end is black hole quencher 1 ( Black Hole Quencher1, BHQ1).

[0054] Among them, HER2(R)-F: AACACCCACCTCTGCTTCGT (SEQ ID NO: 1),

[0055] HER2(R)-R: GCCCACACACTCGTCCTCT (SEQ ID NO: 2),

[0056] HER2(R)-P: TGCCCTGGGACCAGCTCTTTCG (SEQ ID NO: 3).

[0057] 2. Design of primers and probes

[0058] Specific primers GAPDH-F and GAPDH-R were designed for GAPDH gene, and Taqman fluorescent probe GAPDH-FP. The fluorescent reporter group labeled at the 5' end of the Taqman fluorescent probe GAPDH-FP is 6-carboxyfluorescein (6-carboxyfluorescein, FAM), and the quencher group labeled at the 3' end is Black Hole Quench...

Embodiment 2

[0081] Example 2 Construction of HER2 / GAPDH quantitative standards I-IV:

[0082] (1) Amplify HER2 and GAPDH gene fragments

[0083] The PCR reaction solution included 2.5 μL of 10× reaction buffer, 0.5 μL of HER2(R)-F (10 μM), 0.25 μL of HER2(R)-R (10 μM), 0.5 μL of GAPDH-F (10 μM), 0.5 μL of GAPDH-R (10 μM ) 0.25 μL, dNTPs 2.0 μL, and finally make up the reaction system to 19.8 μL with sterile ultrapure water.

[0084] 19.8 μL of PCR reaction solution, 0.2 μL of DNA polymerase, and 5.0 μL of purified human total RNA were used for PCR reaction. The reaction conditions of PCR reaction were: 94°C, 4 minutes; 94°C, 15 seconds; 60°C, 35 seconds, 40 cycles. The amplified products include HER2 and GAPDH gene fragments.

[0085] (2) The amplified product is inserted into the T vector to construct a recombinant plasmid, and the recombinant plasmid is transformed into bacteria and then amplified.

[0086] The obtained DNA sequence was directly ligated to the pSTV28 DNA vector by...

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Abstract

The invention discloses a detection primer combination and a kit for HER2 (human epidermal growth factor receptor 2) gene amplification. The detection primer combination comprises an HER2 primer combination and a GAPDH reference primer combination; each combination comprises upstream and downstream amplification primers of a target gene of the combination and a Taqman fluorescent probe. The kit comprises the detection primer combination, a positive quality control and a negative quality control. By using the detection primer combination and the kit, whether the HER2 gene in patients with breast carcinoma is amplified or not can be detected quickly and simply; compared with the FISH (fluorescent in situ hybridization) method, a method using the detection primer combination and the kit is high in detection sensitivity, low time consumption, generally 1.5 to 2 hours for acquiring reaction results, low in cost, low in false positive ratio, and suitable for large-scale clinical development; thus, quick, effective and accurate detection is achieved for HER2 gene amplification, and timely disease treatment and treatment effect monitoring are guaranteed.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a detection primer set and a kit for HER2 gene amplification. Background technique [0002] Breast cancer is one of the most common cancers in women. Because the initial symptoms of breast cancer are not obvious, it is often malignant when diagnosed, which seriously endangers women's lives. In 2002, the incidence of breast cancer in the world reached 1.2 million. By 2010, this figure exceeded 1.6 million. Meanwhile, breast cancer was responsible for 425,000 female deaths in 2010. China is one of the countries with the fastest-growing incidence of breast cancer. Statistics released by the China Anti-Cancer Association show that the incidence of breast cancer in my country has been increasing at a rate of 3% per year in recent years, becoming the cancer with the fastest-growing mortality rate in cities. The age of onset also showed a gradually younger trend. The age of on...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/166
Inventor 丁朋举贾哲王林海刘沛
Owner 北京鑫诺美迪基因检测技术有限公司
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