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Real-time fluorescence PCR detection kit and detection method for DAB2IP genes

A detection kit and real-time fluorescence technology, which is applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve difficult measurement problems, achieve improved sensitivity, high sensitivity, and reduce false positive interference Effect

Inactive Publication Date: 2011-04-06
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the research on DAB2IP is in the ascendant, and the methods for conventional detection of DAB2IP expression are still mainly based on immunohistochemistry and semi-quantitative PCR. The former is difficult to detect in peripheral blood, and the latter can only play a qualitative role.

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  • Real-time fluorescence PCR detection kit and detection method for DAB2IP genes
  • Real-time fluorescence PCR detection kit and detection method for DAB2IP genes
  • Real-time fluorescence PCR detection kit and detection method for DAB2IP genes

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Embodiment 1

[0044] Example 1: Specific primers, fluorescent probes

[0045] 1. Materials:

[0046] RNA extraction reagents, reverse transcription reagents, and PCR reaction reagents were all purchased from Dalian Bao Bioengineering Co., Ltd.; Taq DNA polymerase was purchased from Promega Company of the United States, and MX3000P quantitative PCR instrument was a product of Agilent-Stratagene Company of the United States.

[0047] 2. Primer and probe design and synthesis:

[0048] Using the DAB2IP gene sequence (registration number NM 032552.2) as a template, use PrimerExpress TM (V2.0, American ABI Company) software analyzes TaqMan primer and probe site, selects the optimal combination therefrom.

[0049] The sequences of PCR primers and probes for detection are as follows:

[0050] Upstream primer: 5′-TGTGAAGTGGACCCAAGCAA-3′

[0051] Downstream primer: 5′-ACGCAGTAGGAGTTGATGATTTTG-3′

[0052] Fluorescent probe: 5′-FAM-CCCGAGCACCAGGGCAACCTC-TAMRA-3′

[0053] Among them, FAM is a fluo...

Embodiment 2

[0055] Embodiment 2: Fluorescent quantitative PCR method detects DAB2IP gene

[0056] 1. Clinical sample testing:

[0057] 5 cases of clinical tumor tissue specimens, the total RNA was extracted with RNA extraction reagent RNAi Plus (Dalian Bao Biology, D19108A), and 1.0ug was taken for reverse transcription reaction, 2×RT buffer, 10.0μL; dNTP (10mM), 1μL; Oligo dT (10uM), 1μL; RNA inhibitor, 0.5μL; RTase, 1μL; total RNA 1μg, made up to 20μL with water. Reverse transcription conditions: 30°C, 10 minutes; 42°C, 30 minutes; 95°C, 5 minutes, take 1ul cDNA as a template, and perform PCR amplification on an Agilent-Stratagene MX3000P quantitative PCR instrument with upstream and downstream primers for detection.

[0058] The composition of the PCR reaction solution is as follows:

[0059] 2×PCR buffer 10.0μL

[0060] Detection upstream primer (10μM) 1μL

[0061] Downstream primer for detection (10μM) 1μL

[0062] Fluorescent probe for detection (10μM) 0.5μL

[0063] DNA polym...

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Abstract

The invention provides a real-time fluorescence PCR detection kit for DAB2IP genes, which mainly comprises a specific primer, a fluorescence probe, PCR buffer solution, a deoxynucleotide triphosphate mixture and DNA polymerase, wherein in the sequences of the specific primer and the fluorescence probe, the upstream primer is 5'-TGTGAAGTGGACCCAAGCAA-3', the downstream primer is 5'-ACGCAGTAGGAGTTGATGATTTTG-3' and the fluorescence probe is 5'-FAM-CCCGAGCACCAGGGCAACCTC-TAMRA-3', wherein the FAM is a fluorescence report group, and the TAMRA is a fluorescence quenching group. The kit has the main advantages that: a method of the invention has high detection sensitivity of DAB21IP genes and good specificity, and reduces the false positive rate of conventional PCR amplification; and the kit can realize quick, accurate and specific detection and analysis of the DAB2IP genes, also can be added with a standard substance for quantitative detection, and can be added with housekeeping genes (such as beta-actin genes and the like) for relative quantitative detection at the same time.

Description

(1) Technical field [0001] The invention relates to a real-time fluorescent PCR detection kit of DAB2IP gene and a detection method thereof. (2) Background technology [0002] Adaptive and self-accelerating growth is the only way for the occurrence and development of malignant tumors. As an important expression network regulator, DAB2IP belongs to the RAS-GTPase activating protein family and has the activity of inhibiting tumor cell proliferation. pathway to inhibit tumor growth and metastasis. DAB2IP can attenuate the expression of oncogenes such as H-RAS, R-RAS and TC21, and is associated with TNF-a-mediated tumor cell apoptosis. As a target gene of EZH2, DAB2IP may be involved in EZH2-involved tumor epigenetic silencing regulation. In addition, DAB2IP can mediate the inactivation of the PI3K-Akt pathway, which is an important tumor cell signaling pathway that regulates tumor proliferation, survival and metastasis. The latest study found that DAB2IP can also block tumor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 方维佳林才照徐农
Owner ZHEJIANG UNIV
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