Huaman plasma DNA quantitative analyser

A technique for quantitative analysis of human plasma

A technique for quantitative analysis of human plasma

CN1811387AInactive Publication Date: 2006-08-02潘世扬
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  • Huaman plasma DNA quantitative analyser
  • Huaman plasma DNA quantitative analyser
  • Huaman plasma DNA quantitative analyser

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0030] 1. Preparation of Exogenous DNA

[0031] Insert the oligonucleotide with A at the 3' end into the vector pMD18T to prepare competent bacteria, and transfer the whole amount of pMD18-T containing the target fragment into 200 μl of competent bacteria; after blue-white screening and colony PCR identification, it is positive Cloning and extraction of plasmid DNA containing the target fragment.

[0032] Restriction endonuclease Hind II digests the plasmid internal reference to linearity: After digestion, dilute the linear cloned plasmid DNA internal reference to 1×10 3 copies / μl.

[0033] 2. Plasma Separation and Storage

[0034] with EDTA-K 2 Extract 3ml of human peripheral blood from a 5ml sterile plastic anticoagulant centrifuge tube with a cover, and centrifuge at 2000r / min for 20min within 4h at room temperature to collect plasma; centrifuge again at 3000r / min for 15min to obtain blood cell-free plasma, take 0.5ml and collect To which was added a concentration of 1×...

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Abstract

The present invention relates to a quantitative detection analysis method of human plasma DNA level. Said method includes the following steps: selecting artificially-synthetic exogenous DNA series whose extraction efficiency is identical to or similar to that of human plasma DNA, but they are non-homologous, selecting housekeeping gene beta actin as determination gene, inserting artificially-synthetic exogenous DNA into carrier pMD18T and placing it in the bacteria to make culture, making blue and white spot screening, colony PCR identification, enzyme-cutting into linearity, placing the above-mentioned material into plasma, adopting double fluorescent quantitative gene amplification, fluorescent groups are respectively FAM and HEX, extracting known exogenous positive plasmid recovery efficiency, using computation formula to make computation so as to obtain the quantity of plasma DNA.

Description

technical field [0001] The invention relates to a DNA measurement method, in particular to a quantitative detection and analysis method for plasma DNA level. Background technique [0002] Human plasma DNA refers to free extracellular DNA (cell free DNA), also known as circulating DNA, which is always at a relatively constant level and maintains a balance. Only when the human body suffers from various diseases, such as: body inflammation caused by infection, large area tissue damage caused by trauma, failure of tissues and organs, autoimmune diseases and tumors, etc. due to a large number of cell death, nucleic acid is released from the cells This balance will be broken only when a large amount of internal organs enter the blood of the peripheral circulation, resulting in a substantial increase in human plasma DNA levels and abnormal DNA quality. [0003] Therefore, the quantitative detection and analysis of plasma DNA can be used for the diagnosis and severity assessment of...

Claims

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Application Information

Patent Timeline
02 Aug 2006
Publication
CN1811387A
IPC
G01N21/64; C12Q1/68
Inventors
潘世扬; 黄珮珺