Fluorescent quantitative PCR diagnostic kit for rapidly detecting HER-2 mRNA

A HER-2, fluorescence quantitative technology, applied in fluorescence/phosphorescence, material excitation analysis, etc., to achieve simple and safe operation, low false positives, and good specificity

Inactive Publication Date: 2012-05-23
WUHAN KANGYUAN BIOLOGICAL MEDICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the US Food and Drug Administration (FDA) has approved the use of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) techniques to assess HER2 levels, but whether this method is the best for detecting HER2 status is still controversial.

Method used

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Embodiment Construction

[0017] The present invention will be further described below in conjunction with a specific embodiment. This embodiment is only used to illustrate the present invention and is not intended to limit the scope of protection claimed by the present invention.

[0018] (1) The kit of the present invention consists of the following:

[0019] ①Trizol

[0020] ② First strand cDNA synthesis kit (RT-PCR)

[0021] ③ Primers: including the target gene HER-2 and the internal reference gene β-actin, as follows:

[0022] ④ Upstream: 5′-GACCGGAGGCTGACCAGT-3′;

[0023] ⑤Downstream: 5′-GGCAAGGCTGGCATGCGG-3′;

[0024] ⑥Beta-actin gene primer sequence:

[0025] ⑦Upstream: 5'-GAAGATGGTGATGGGATTTC-3';

[0026] Downstream: 5'-GAAGGTGAAGGTCGGAGTC-3';

[0027] ⑧Fluorescent probe: FAM 5'-GGCCCGCTGCCCCAGCGGTG-3'TAMRA;

[0028] FAM5'-ACCACCACGGCCGAGCGG-3'TAMRA

[0029] ⑨PCR reaction solution: 1×PCR buffer, 0.3-0.5pmol / μL primer, 0.1-0.3pmol / μL probe, 2.5-4.0mM Mg2+, 1-2U Taq enzyme, 0.2-0.4mM dN...

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Abstract

The invention discloses a fluorescent quantitative PCR diagnostic kit for rapidly detecting HER-2 mRNA, and relates to an oncogene detection technology. The fluorescent quantitative PCR diagnostic kit is composed of a first chain cDNA synthesis kit, a PCR reaction solution and a lightcycler PCR instrument. The first chain cDNA synthesis kit comprises MgCl2, a reverse transcriptase buffer, dNTP, a RNA enzyme inhibitor, Oligo (dT) 15, AMV reverse transcriptase and DEPC water, and synthesis of HER-2, a reference gene primer and a Taqman fluorescent probe; the primer is divided into an upstream primer and a downstream primer. The invention has the advantages of good specificity, sensitivity and repeatability, accurate quantification, rapidity and convenience, is suitable for specifically qualitative and quantitative determination of HER-2 gene at early stage, and provides better reference for the chemotherapy and prognosis for the clinical breast cancer.

Description

technical field [0001] The invention relates to a gene diagnosis kit for rapidly detecting the expression level of HER-2 mRNA in breast cancer and its application. Background technique [0002] The proto-oncogene HER-2 is also called c-erbB-2 or HER-2 / neu. The HER-2 gene is located on human chromosome 17q21-22 and encodes a cell membrane glycoprotein with tyrosine kinase activity. HER-2 molecules exist in the form of monomers on the cell surface, and are basically inactive at this time. When they bind to ligands in the extracellular functional area to form stable receptor dimers, they can generate activation signals and promote the overexpression of EGFR on the cell membrane. , so as to participate in the regulation of cell growth, reproduction and division and control the growth of tumors. [0003] Studies have shown that HER-2 can be used as a prognostic indicator for breast cancer. Clinically, HER-2-positive breast cancer patients, especially those with overexpression, o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 童永清李艳
Owner WUHAN KANGYUAN BIOLOGICAL MEDICAL TECH
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