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Kit for detecting high mobility group chicken protein B1 by real-time fluorescent quantitative reverse-transcription polymerase chain reaction

A reverse transcription polymerase, real-time fluorescence quantitative technology, applied in fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc. The effect of repeated detection methods

Inactive Publication Date: 2012-12-05
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on chHMGB1 is still in the initial stage, and various methods for detecting the expression of chHMGB1 are still lacking.

Method used

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  • Kit for detecting high mobility group chicken protein B1 by real-time fluorescent quantitative reverse-transcription polymerase chain reaction
  • Kit for detecting high mobility group chicken protein B1 by real-time fluorescent quantitative reverse-transcription polymerase chain reaction
  • Kit for detecting high mobility group chicken protein B1 by real-time fluorescent quantitative reverse-transcription polymerase chain reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Fluorescent quantitative RT-PCR method to detect the expression of chHMGB1

[0036] 1. Materials:

[0037] DNase Ⅰ was purchased from Fermentas Company. The reverse transcription kit was purchased from Dalian TaKaRa Company. T vector ligase was purchased from Promega. dNTPs were purchased from Shanghai Sangon Bioengineering Co., Ltd. The total RNA extraction kit was produced by Hangzhou Axygen. Premix Ex Taq TM Ⅱ is the product of TaKaRa Company. Others are domestic analytical reagents.

[0038] 2. Primer and probe design and synthesis:

[0039] Using the full-length cDNA sequence of chHMGB1 (GenBank accession number Y17968) as a template, use PrimerExpress TM (V3.0, American ABI Company) software analyzes and designs primers, and selects the best primers based on the simultaneous consideration of the chHMGB1 genomic DNA sequence.

[0040] The PCR upstream primer sequence for detection is: 5'-CCAAATGCACCGAAGAGG-3' (SEQ ID NO.1), and the downstream pri...

Embodiment 2

[0044] Example 2 Fluorescent quantitative RT-PCR method to detect the expression of chHMGB1 in the tissues and organs of 1-day-old chicks

[0045] 1. Specimen testing:

[0046] The tissues and organs of 1-day-old chicks were ground with liquid nitrogen, and the total RNA was extracted with the Axgen Total RNA Extraction Kit. Take 1 μg of RNA, and Oligo(dT) in a total reaction volume of 20 μl 15-18 After performing reverse transcription as primers, use the upstream and downstream primers for detection to carry out PCR amplification on the 7500 fluorescent quantitative PCR instrument of ABI Company. 40 cycles of amplification were performed in 30 seconds, and finally extended at 72°C for 10 minutes and placed at 4°C. At the same time, add standard substance to test for standard curve. The measurement results were processed by software to calculate the chHMGB1 expression level of the test specimens according to the standard curve.

[0047] 2. Sample test results

[0048] For ...

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Abstract

The invention belongs to biotechnology field, and relates to quantitative RT(reverse transcription)-PCR (polymerase chain reaction) detection kit, in particularly to a kit for detecting high mobility group chicken protein B1 by real-time fluorescent quantitative reverse-transcription polymerase chain reaction. The kit comprises RT reaction liquid, reverse transcriptase of Moroni rat leukoviruses, RNA enzyme inhibitor, PCR liquid, heat-resisting DNA polymerase, standard substance and reference substance. The kit can obtain cDNA by RT of mRNA sample, and work with real-time fluorescence quantification PCR detection technology to precisely quantify the mRNA expression index of high mobility group chicken protein B1 in the detecting sample. The kit can be used to analyze the expressing of HMGB1 in tissue and viscera of the chicken and also used to detecting the change of the expression level of the HMGB1 in chicken cells in different conditions. The invention provides a quick, accurate and repeatable detection method for the expression level of HMGB1 in chicken cells.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a quantitative RT-PCR detection kit, in particular to a real-time fluorescence quantitative RT-PCR detection kit for chicken high mobility group protein B1. Background technique [0002] High mobility group protein (HMG) was first discovered by Johns in the calf thymus in the 1970s. It is a non-histone chromosome-binding protein that exists in almost all eukaryotic cells because of its small molecular weight (<30kD) , which migrate rapidly in polyacrylamide gel electrophoresis and are named high mobility group proteins (HMG proteins). According to molecular mass and DNA binding properties, HMG family proteins are divided into HMGA, HMGB, and HMGN families; HMGB is further divided into HMGB1 and HMGB2. HMGB1 is highly conserved during evolution, with 99% homology among all mammals. The HMGB1 molecule includes three functional regions: the A box (1aa~79aa) and the B box (89a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 钱琨秦爱建朱明月张娜金文杰邵红霞
Owner YANGZHOU UNIV
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