Nucleotide sequence and kit for HER-2 gene copy number amplification detection, and application of kit

A gene copy number and gene amplification technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of technical difficulty and high cost, difficult result analysis, long detection process, etc., to achieve Good effect, simple preparation, high degree of automation

Active Publication Date: 2018-02-27
昆山迪安医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, FISH relies on the quality of probes produced and the quality of pre-production samples, and is also interfered by polyploidy of chromosome 17. The detection process is long, the operation is complicated, the result analysis is difficult, and the technical difficulty and cost are high.

Method used

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  • Nucleotide sequence and kit for HER-2 gene copy number amplification detection, and application of kit
  • Nucleotide sequence and kit for HER-2 gene copy number amplification detection, and application of kit
  • Nucleotide sequence and kit for HER-2 gene copy number amplification detection, and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Primers and probes for detecting HER-2 and human gene 21-TFF3 were synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd., and the sequences are as follows:

[0032] Primers for amplifying HER-2:

[0033]F1 (SEQ ID NO: 1): 5'-tcactcatatcctcctctttctgc-3';

[0034] R1 (SEQ ID NO: 2): 5'-aattttcacattctccccatcag-3';

[0035] Probes to detect HER-2:

[0036] P HER-2 (SEQ ID NO: 3): 5'-cagggcatctggatc-3';

[0037] Wherein, the 5' end of the HER-2 probe P HER-2 (SEQ ID NO: 3) sequence is marked with a FAM fluorescent group, and the 3' end is marked with an MGB quencher group.

[0038] Primers for amplifying the human 21-TFF3-S gene:

[0039] F3 (SEQ ID NO: 4): 5'-gcctggatcttgcctggag-3';

[0040] R3 (SEQ ID NO: 5): 5'-aaatcctaatgcttggggcac-3';

[0041] Probes for detection of human 21-TFF3 gene:

[0042] P21-TF F3 (SEQ ID NO: 6): 5'-ccagttacatcagggca-3';

[0043] Wherein, the 5' end of the human 21-TFF3 gene probe P21-TFF3 (SEQ ID NO: 6) sequence is marked...

Embodiment 2

[0047] Example 2: Preparation method of HER-2 gene amplification kit.

[0048] (1) PCR reaction solution: 2*supermix (purchased from Bio-rad Company in the United States, item number 186-3026), which is a 2*PCR reaction premix, which contains DNA polymerase, Mg 2+ , PCR reaction buffer, dATP, dCTP, dTTP and dGTP and other components, stored at -20°C;

[0049] (2) Primer-probe premix solution: Dissolve the primers and probes shown in SEQ ID NO: 1-8 in double distilled water, the concentration of each primer is 100 μM, and the concentration of each probe is 10 μM; 1 The mother solutions of ~3 nucleic acid sequences are mixed at a ratio of 9:9:25; the mother solutions of 4~6 nucleic acid sequences are mixed at a ratio of 9:9:25; the mother solutions of 6~8 nucleic acid sequences are mixed at a ratio of 25:9:9 Proportionally mixed, the final primer / probe concentration in the reaction system is 900 / 250nM. Store at -20°C;

[0050] (3) Positive control: Formalin-soaked paraffin ti...

Embodiment 3

[0052] Embodiment 3: detection method.

[0053] Instruments: Eppendorf Mastercycler pro S qualitative PCR instrument, Bio-rad QX200 droplet digital PCR system (including droplet generator, sealing device, droplet reader), BECKMAN 22R desktop micro-refrigerated centrifuge, WH-866 vortex oscillator (Taicang Hualida), low-speed plate centrifuge (Anhui Zhongjia).

[0054] 1. Preparation of HER-2 sample, positive control, and negative control DNA template: use QIAamp DNAFFPE Tissue Kit from QIAGEN Company (Cat. No. 157017130), and operate according to the kit instructions.

[0055] 2. Using the nucleic acid obtained in step (1) as a template, use 3 pairs of specific primers and 2 specific fluorescent probes to perform amplification detection of HER-2 and 21-TFF3 genes, specifically including the following steps;

[0056] (1) Preparation of PCR reaction solution: Take out the components of the kit from the -20°C refrigerator, thaw at room temperature, and put them on an ice box fo...

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Abstract

The invention discloses a nucleotide sequence and a kit for detecting a human epidermal growth factor receptor-2 (HER-2), which belong to the technical field of molecular diagnosis. A primer and a probe for detecting the HER-2 provided by the invention can be used for specifically amplifying and detecting amplification of an HER-2 gene. The invention also discloses the detection kit for detectingthe HER-2 gene based on a microdroplet type PCR (Polymerase Chain Reaction) system, so that the amplification of the HER-2 gene can be quickly, sensitively and accurately detected. The nucleotide sequence and the kit provided by the invention have the advantages of simplicity and convenience in operation, high specificity, high sensibility, low cost, high throughput and the like, can be used for quickly detecting clinic HER-2 gene amplification, and provide references for diagnosis and treatment of the clinic human epidermal growth factor receptor-2.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a nucleotide sequence for detection of HER-2 gene copy number amplification, a kit and an application thereof. Background technique [0002] Human epidermal growth factor receptor 2 (human epidermal growth factor receptor-2, HER-2) gene is located on chromosome 17q12-q21, and its encoded product is 185kD transmembrane protamine p185, which consists of 1255 amino acids, and positions 720-987 belong to Tyrosine kinase domain. HER-2 / neu protein is a transmembrane glycoprotein with tyrosine protein kinase activity, a member of the epidermal growth factor receptor (EGFR) family, and highly homologous to EGFR. Studies have confirmed that HER-2-mediated signal transduction is related to the formation, proliferation, metastasis, invasion and resistance to radiotherapy and chemotherapy of various tumors. In the past, most of its related research focused on breast...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6886C12Q2600/118C12Q2600/156C12Q2563/159
Inventor 任绪义虞闰六宣文静周磊罗英
Owner 昆山迪安医学检验实验室有限公司
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