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Detection method of real-time fluorescence quantification (PCR) for DSPP (dentin sialophosphoprotein) genes in incisors of guinea pig

A real-time fluorescence quantitative and detection method technology, applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of incomplete function of gene-encoded protein, no DSPP gene, dentin hypoplasia, etc., to achieve specific Strong sex, high sensitivity, low pollution effect

Inactive Publication Date: 2015-03-25
赤峰市农牧科学研究院
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  • Application Information

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Problems solved by technology

DSPP mutation can lead to incomplete function of the protein encoded by the gene, which affects tooth mineralization and dentin hypoplasia
There is no report on the real-time fluorescent quantitative PCR detection method of DSPP gene in guinea pig incisors

Method used

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  • Detection method of real-time fluorescence quantification (PCR) for DSPP (dentin sialophosphoprotein) genes in incisors of guinea pig
  • Detection method of real-time fluorescence quantification (PCR) for DSPP (dentin sialophosphoprotein) genes in incisors of guinea pig
  • Detection method of real-time fluorescence quantification (PCR) for DSPP (dentin sialophosphoprotein) genes in incisors of guinea pig

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Embodiment

[0033] A. Sample preparation: all related instruments and consumables are sterilized and RNase-removed; two pairs of upper and lower incisors of guinea pigs are quickly extracted, and the attached gingival tissue is removed as much as possible with a scalpel, and sterilized saline at 4°C is used repeatedly Rinse and blot dry with sterilized filter paper; quickly cut the teeth into small pieces with sterilized rongeurs, put them in liquid nitrogen for cryogenic embrittlement treatment for 48 h;

[0034] B. Design primers: Design corresponding primers according to the mRNA sequences of the guinea pig β-actin gene (accession NO. AF508792) and the rat DSPP gene (accession NO. NM_012790) in Genbank.

[0035] The primers used for β-actin gene amplification are:

[0036] Upstream primer: 5'-GGATGCAGAAGGAGATCACG-3'

[0037] Downstream primer: 5'-ACATCTGCTGGAAGGTGGAG-3'

[0038] The primers used for DSPP gene amplification are:

[0039] Upstream primer: 5'-CGAGTCGATAGCCGTAGGAG-3'

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Abstract

The invention discloses a detection method of real-time fluorescence quantification PCR for DSPP (dentin sialophosphoprotein) genes in incisors of a guinea pig. The detection method comprises the following steps of: A. preparing a sample: pulling out teeth so as to carry out low temperature freezing embrittlement treatment for 24-48h; B. designing a primer; C. extracting the total RNA (ribonucleic acid); D. establishing a reaction system; E. optimizing a reaction condition; F. manufacturing a standard curve; and G. carrying out fluorescent quantitative analysis. The method is used for detecting the real-time fluorescence quantification PCR for the DSPP genes in the incisors. The detection method provided by the invention has the advantages that the operation is simple, the pollution is slight, the susceptibility is high, the specificity is strong, the repeatability is good, and the accuracy is high.

Description

technical field [0001] The invention relates to a detection method of fluorescence quantitative PCR, in particular to a detection method of real-time fluorescence quantitative PCR of DSPP gene in guinea pig incisors. Background technique [0002] Dentin sialophosphoprotein gene (Dentin Sialophosphoprotein, DSPP) encodes two proteins, Dentin sialoprotein (DSP) and Dentin phosphoprotein (DPP). The two are two fragments of different sizes and different biological functions produced by cutting the expression product of the same transcript. DPP and DSP are important non-collagen proteins secreted by odontoblasts, they participate in the epithelial-mesenchymal interaction, promote the directed differentiation of odontoblasts, and resist proteolysis by initiating crystal nucleation, regulating crystal morphology and growth rate Enzyme degradation function, etc., so as to ensure the calcification of dentin matrix. DSP is a glycoprotein, which accounts for about 5% to 8% of the tot...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 韩天龙李志明王敏赵瑞霞黄国成李清泉
Owner 赤峰市农牧科学研究院