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Kit for detecting HER2 gene amplification by digital PCR

A gene amplification and digital application technology, applied in the field of HER2 gene amplification detection reagents, can solve the problems of inconvenient observation results, limited professional knowledge and laboratory conditions, and cumbersome operations, and achieve the effect of reducing human interference and data amplification. Good, reducing the effect of false positives

Inactive Publication Date: 2020-01-10
宁波胤瑞生物医学仪器有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • 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.
Currently, FISH and IHC methods are used, but FISH is expensive, has a long detection period, high patient fees, limitations of tester expertise and laboratory conditions, which is not conducive to wide application
IHC also has disadvantages such as cumbersome operation and inconvenient observation results, so this invention is proposed

Method used

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  • Kit for detecting HER2 gene amplification by digital PCR
  • Kit for detecting HER2 gene amplification by digital PCR
  • Kit for detecting HER2 gene amplification by digital PCR

Examples

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Effect test

Embodiment 1

[0073] A kit for detecting HER2 gene amplification by digital PCR, which includes a primer-probe premix, which includes 1) upstream and downstream primers for detecting HER2 genes, and fluorescent probes for detecting HER2 genes; ) detecting the upstream and downstream primers of the human FAS gene, and the fluorescent probe for detecting the human FAS gene; 3) detecting the upstream and downstream primers of the human ACTB gene, and the fluorescent probe for detecting the human ACTB gene;

[0074] Wherein, 1) primers and probes of the HER2 gene include the following sequences:

[0075] Upstream primer: 5'-ACTTAATTGGCATTCTACTCCC-3'SEQ ID NO:1;

[0076] Downstream primer: 5'-ATTGGAGACACGGCCTC-3'SEQ ID NO:2;

[0077] Probe: 5'-GGCGCAACGCTGAGCAGCTGG-3'SEQ ID NO:3;

[0078] 2) The upstream and downstream primers and probes for detecting the human FAS gene include the following sequences:

[0079] Upstream primer: 5'-CGCTTATATAATGGTGCTCGTT-3'SEQ ID NO:4

[0080] Downstream prim...

Embodiment 2

[0093] The method for detecting HER2 gene amplification using the above kit comprises the following steps:

[0094] (1) Preparation of samples to be tested

[0095] The applicable specimen type of the present invention is a frozen tissue that is frozen immediately after surgery and stored at -20°C, or a paraffin-embedded tissue that is routinely processed;

[0096] (2) Preparation of DNA template

[0097] Use paraffin-embedded tissue, use Qiagen DNeasy Blood and Tissue Kit (Cat.No69504), operate according to the kit instructions, and finally collect 50 μL DNA solution, directly detect or store at -20°C.

[0098] (3) Preparation of reaction system

[0099] Mix the master mix with primers, probes and templates according to the ratios in the table below, and carry out digital PCR amplification reaction.

[0100]

[0101]

[0102] (4) Detection process

[0103] Set the thermal cycle temperature according to the table below, and select the detection channel suitable for d...

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Abstract

The invention belongs to the technical field of HER2 gene amplification detection reagents, and particularly relates to a kit for detecting HER2 gene amplification by digital PCR. The kit comprises aprimer probe premixed solution, the primer probe premixed solution comprises (1) upstream and downstream primers for detecting an HER2 gene and a fluorescent probe for detecting the HER2 gene; (2) upstream and downstream primers for detecting a human FAS gene, and a fluorescent probe for detecting the human FAS gene; and (3) upstream and downstream primers for detecting a human ACTB gene, and a fluorescence probe for detecting the human ACTB gene. The kit for detecting the HER2 gene amplification provided by the invention can conduct absolute quantification compared with real-time fluorescentquantification PCR, and a detection lower limit is obviously lower than the fluorescent quantification PCR. Moreover, a primer probe combination of the kit has high specificity and better detection effect.

Description

technical field [0001] The invention belongs to the technical field of HER2 gene amplification detection reagents, in particular to a kit for detecting HER2 gene amplification by digital PCR. Background technique [0002] The human HER-2 / neu gene (Her-2 gene) gene is located on chromosome 17q21, and its encoded product is a 185kD transmembrane protamine p185, which consists of 1255 amino acids, and the 720-987 position belongs to the tyrosine kinase region. Her-2 / neu protein is a transmembrane glycoprotein with tyrosine protein kinase activity and is a member of the EGFR family. Overexpression of human epidermal growth factor receptor 2 (HER2, ERBB2) is closely related to the progression of many epithelial cancers. Tumors with high expression of HER2 show strong ability of metastasis and invasion, poor sensitivity to chemotherapy, and easy recurrence. The expression level of HER2 in normal cells is very low, but the expression level is very high during embryonic developmen...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6851C12Q1/6886C12Q2600/158C12Q2600/166C12Q2531/113C12Q2521/525C12Q2563/107C12Q2545/101
Inventor 金鑫浩刘一博隋硕任鲁风张未来俞育德于军
Owner 宁波胤瑞生物医学仪器有限责任公司
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