Application of hTERT (human Telomerase Reverse Transcriptase) gene serving as reference gene in direct detection of content of cfDNA (cell-free Deoxyribonucleic Acid) in blood plasma by utilizing qPCR (quantitative Polymerase Chain Reaction)

An internal reference gene and gene technology, applied in the field of hTERT gene, can solve problems such as difficult operation and inability to reflect the true level of plasma free DNA, and achieve the effect of reducing background noise

Inactive Publication Date: 2018-05-01
北京中源维康基因科技有限公司
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Problems solved by technology

[0004] Existing technologies include qPCR method, bDNA technology method, and fluorescent dye method, each ...

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  • Application of hTERT (human Telomerase Reverse Transcriptase) gene serving as reference gene in direct detection of content of cfDNA (cell-free Deoxyribonucleic Acid) in blood plasma by utilizing qPCR (quantitative Polymerase Chain Reaction)
  • Application of hTERT (human Telomerase Reverse Transcriptase) gene serving as reference gene in direct detection of content of cfDNA (cell-free Deoxyribonucleic Acid) in blood plasma by utilizing qPCR (quantitative Polymerase Chain Reaction)
  • Application of hTERT (human Telomerase Reverse Transcriptase) gene serving as reference gene in direct detection of content of cfDNA (cell-free Deoxyribonucleic Acid) in blood plasma by utilizing qPCR (quantitative Polymerase Chain Reaction)

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Embodiment Construction

[0018] Below in conjunction with accompanying drawing and specific embodiment the present invention is described in further detail:

[0019] 1. Preparation and dilution of the plasma to be tested: after separating the plasma from the blood, add protease (PK) for digestion; then remove the digested plasma and dilute it at a ratio of 1:40

[0020] 2 Preparation of qPCR reaction system

[0021]

[0022] mix (contains 10×PCR buffer 2μl, SYBR Green saturating dye 1μl, 5U / μl Taq DNApolymerase 0.2μl, 15mM MgCl 22μl, 10mM dNTP 0.5μl and ddH2O 7.3μl, wherein 10×PCRbuffer contains 10-40mM Tris-Cl, 50-200mM potassium chloride , 0-5.0mM dithiothreitol (DTT), 0-1.0mM ethylenediaminetetraacetic acid disodium calcium (EDTA), 0-20vol% ethylphenyl polyethylene glycol (Nonidet) P-40, 0-2.0vol% Tween 20 (Tween20), 30-70vol% glycerin and stabilizer (pH7.0-10.0))

[0023] 3 Set up the qPCR reaction program

[0024]

[0025] 4. Analyze quantitative data: confirm that the amplification effi...

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Abstract

The invention discloses application of an hTERT (human Telomerase Reverse Transcriptase) gene serving as a reference gene in direct detection of the content of cfDNA (cell-free Deoxyribonucleic Acid)in blood plasma by utilizing qPCR (quantitative Polymerase Chain Reaction). According to the application disclosed by the invention, the aim of quantifying the cfDNA is realized through detecting a house-keeping gene in DNA or certain non-coding repeated sequences which appear stably. Before the qPCR is carried out, the blood plasma is treated by utilizing a prepared buffering solution and prepared proteinase K to remove protein; special polymerase (velocity polymerase) which is used for difficult template amplification is added into a reaction mixture and the polymerase can be used for extending 1kb of basic group every 10S; after the blood plasma is diluted according to the ratio of 1 to 40, other treatment is not needed; then the blood plasma is directly added into a reaction system andis subjected to the qPCR to determine the concentration of the cfDNA in the blood plasma; the cfDNA is quantified through amplifying target sequences with two lengths. By adopting the application, error mutation caused by PCR and sequencing can be effectively removed and background noises in a detection process are reduced. With regard to 0.1 percent of detection limit, the sensitivity can reach90 percent or more and the specificity reaches 99.9 percent.

Description

technical field [0001] The present invention relates to the use of hTERT gene, especially the use of hTERT gene as an internal reference gene in the direct detection of cfDNA content in plasma by qPCR. Background technique [0002] In recent years, the relationship between telomerase and the occurrence and development of cancer has become one of the hot spots in the field of cancer research. Replication of somatic cells is mostly limited by telomere depletion, but tumor cells can maintain telomere length through telomerase reverse transcriptase (hTERE). Studies have shown that there is a strong correlation between telomerase activity and hTERE expression, and hTERE is a key factor for telomerase activation. In most tumor cells, the expression of hTERE is mainly expressed at the transcriptional level. After the analysis of the deletion of the hTERE promoter sequence, the core region of the hTERT promoter contains multiple transcription factor binding sites, such as sp1, c2my...

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6851C12Q1/6848
CPCC12Q1/6886C12Q1/6848C12Q1/6851C12Q2600/166C12Q2531/113C12Q2545/101C12Q2549/119
Inventor 李同恩罗玺师鸿翔郑康朱明宇周茜刘春柳赵圆圆张文会王红飞刘媛张振宇赵鹏王秉孝朱兵
Owner 北京中源维康基因科技有限公司
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