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Nucleic acid combination for detecting Her2 gene, kit and application

A kit and gene technology, applied in the field of detection of HER2 gene nucleic acid combinations, can solve the problems of false negatives, cumbersomeness, and expensive probe costs of FISH results, and achieve the effects of reducing false negatives, fast analysis speed, and improving accuracy

Active Publication Date: 2017-04-26
领航医学科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, FISH detection takes a long time, the cost of probes is relatively expensive, and professionally trained technicians are required to perform cumbersome operations. In addition, some studies have shown that polysomy of chromosome 17 can lead to false negative results of FISH
[0004] Current detection generally requires invasive tissue biopsy, which is not easy to obtain repeatedly

Method used

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  • Nucleic acid combination for detecting Her2 gene, kit and application
  • Nucleic acid combination for detecting Her2 gene, kit and application
  • Nucleic acid combination for detecting Her2 gene, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The primers and probes were designed for the human Her2 gene sequence. A total of 3 pairs of primers were designed and synthesized, and a probe was designed correspondingly for each pair of primers. The sequences are shown in Table 3.

[0048] table 3

[0049]

[0050] The size of the fragment amplified by the primer pair of Her2-1 was 155 bp, and the sequence of the amplified fragment was: ACAACCAAGTGAGGCAGGTCGTATTGTTCAGCGGGTCTCCACAACCAAGTGAGGCAGGTCccactgcagaggctgcggattgtgcgaggcacccagctctttgaggacaactatgccctggccgtgctagacaatGGAGACCCGCTGAACAATAC.

[0051] The Her2-2 primer pair amplifies the fragment with a size of 125 bp, and the sequence of the amplified fragment is: TCTTCCTCTCCCTACATCGGGTCCAAGTGAACCAGGGAATCTTCCTCTCCCTACATCGGccccacctgtccccaccccctccagcccacagccatgcccacagccagTTCCCTGGTTCACTTGGAC.

[0052] The size of the fragment amplified by the Her2-3 primer pair was 111 bp, and the sequence of the amplified fragment was: GGGCTCTTTGCAGGTCTCTCCCAGCAAGAGTCCCCATCCTAGGGGCT...

Embodiment 2

[0068] The steps of digital PCR detection are as follows:

[0069] (1) Sample preparation: The source of sample DNA can be blood or neutral formalin-fixed paraffin-embedded tissue sample sections. The DNA sample can be properly diluted to ensure that the sample signal copy number is between 200 copies / μL-2000 copies / μL.

[0070] (2) Prepare the PCR reaction solution according to the following ratio: 2 × PCR master mix (using Life Technologies company's 3D Digital PCR Master Mix V2, catalog number: A26358), primers, probes and sample DNA, made up to a final volume of 15 μL with distilled water to prepare a digital PCR mixture. The content of each primer is 900nM, and the content of each probe is 250nM.

[0071] (3) Brush the prepared 15 μL digital PCR mixture onto the chip.

[0072] (4) Put the chip into a PCR machine, and perform amplification according to the following conditions: pre-denaturation at 95°C for 10 minutes; denaturation at 95°C for 15 seconds, annealing and ...

Embodiment 3

[0075] Screening of primers and probes.

[0076] For genomic DNA, a genome is about 3×10 9 bp, the corresponding molecular mass is 3pg, then the copy number of 1ng genomic DNA is about 333. When doing digital PCR, take 1 ng of normal human blood DNA, make 15 μL of reaction solution, and brush it into the chip. Theoretically, the copy number of the target gene (Her2) and the internal reference gene should be 333, but the digital PCR finally shows FAM (Her2 gene) and VIC (internal reference gene) are expressed in copies / μL, 333 copies / 15 μL≈22 copies / μL. The Her2 gene of normal people is not overexpressed, so the copy number ratio of Her2 to the internal control (FAM / VIC) is theoretically 1:1.

[0077] Screening process:

[0078] Detection is carried out according to the detection method in Example 2.

[0079] Template: 1ng DNA extracted from normal human blood

[0080] Primer probe: the following 15 combinations (respectively Her2-1 / RPPH1, Her2-1 / CEP17, Her2-1 / CEP17-1, Her...

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Abstract

The invention discloses a nucleic acid combination for detecting Her2 (human epidermal growth factor receptor-2) gene, a kit and application. The nucleic acid combination comprises two pairs of Her2 gene primers and two primers of internal reference primers. According to the invention, two pairs of primers and two pairs of internal references are employed to detect Her2 gene by digital PCR method, absolute quantification can be performed on Her2 gene, full automatic result interpretation can be carried out by means of software, and the analysis speed is fast. Use of two pairs of primers is conducive to enhancing the accuracy of a detection result, especially for a blood sample with low DNA content in itself. Simultaneous use of two pairs of internal reference primers reduces the possibility of false negative and false positive.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a nucleic acid combination, kit and application for detecting HER2 gene. Background technique [0002] Human epidermal growth factor receptor 2 (human epidermal growth factor receptor-2, Her2) gene, located on chromosome 17q21, encodes a transmembrane receptor-like protein with a relative molecular mass of 185KD and has tyrosine kinase activity. The receptor protein interacts with Her2 family members and their ligands to regulate cell growth, differentiation and proliferation through intercellular signal transduction. Studies have found that about 25%-30% of invasive breast cancer and 80% of gastric cancer patients have Her2 gene amplification or protein overexpression. In addition, breast cancer, ovarian cancer, endometrial cancer, lung cancer, etc. all have the occurrence of Her2 overexpression. Herceptin is an immunotherapy drug approved for the first...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6886C12Q2531/113C12Q2545/101C12Q2600/118C12Q2600/166
Inventor 张菁张顺宋小慧
Owner 领航医学科技(深圳)有限公司
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