Process of micro circulating DNA by quantitative testing

A quantitative detection method and quantitative detection technology, applied in the field of medical testing, can solve problems such as complicated operations, restrictions on the development of similar research, unacceptable prices, etc., and achieve the effect of cheap, universal and convenient use

Inactive Publication Date: 2005-01-05
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because radioimmunoassay involves isotopes and is complicated to operate, it has been basically eliminated at present; the price of fluorescence spectrophotometer and quantitative PCR instrument is more than 100,000 yuan, which is too expensive for some small scientific research institutions and clinical testing departments; Dipstick produced by Invitrogen TM The price of the kit for 50 people is about 3,000 yuan. If it is popularized, the price is unacceptable, which limits the development of similar research in my country to a large extent.

Method used

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  • Process of micro circulating DNA by quantitative testing
  • Process of micro circulating DNA by quantitative testing
  • Process of micro circulating DNA by quantitative testing

Examples

Experimental program
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Effect test

Embodiment 1

[0019] 1. Serum / plasma separation and storage,

[0020] Take 2ml~3ml of peripheral venous blood, put it in a test tube or an anticoagulant tube, let it stand at 10°C~20°C for 4 hours, and centrifuge at 3000 rpm for 10 minutes. Take the upper serum and centrifuge again at the same speed for 10 minutes, aspirate the upper serum to obtain serum or plasma completely free of blood cell components, which can be used directly for detection or stored at -70°C until use.

[0021] 2. Extract free DNA in serum / plasma, and extract serum DNA with "Trace Blood Genome Extraction Kit" (Shanghai Huashun Bioengineering Co., Ltd.). Take 500μl-1000μl of serum, add 2 times the volume of lysate DL and proteinase K, the final concentration is 100μg / ml-150μg / ml, mix well, put it in a 65℃ water bath for 15 minutes to 1 hour, and mix it upside down during the period Several times until the liquid is clear and not viscous. Then add 2 times the volume of serum / plasma isopropanol, mix upside down and ce...

Embodiment 2

[0023] Embodiment 2 formulate standard curve

[0024] Accurately pipette 1 μl of fluorescent dye SYBR Green I (Molecular Probes, USA), add 3000 μl of sterile triple-distilled water, mix well and make a 1:3000 dilution for later use. Take the plasmid DNA whose known concentration is 10ng / μl (the standard amount of DNA comes from the Dipstick of Invitrogen Company in the United States). TM kit), diluted 5 times to a concentration of 2ng / μl for use. Take out the ultraviolet transmission plate of the ultraviolet and visible analysis device of Furi Technology Co., Ltd., spread a transparent film, and spot the sample in the center of the film. Add 10μl triple-distilled water to the first point, add 1μl of 2ng / μl DNA and 9μl triple-distilled water to the second point, add 2μl of 2ng / μl DNA and 8μl triple-distilled water to the third point, add 2ng / μl DNA to the fourth point 3 μl and 7 μl triple-distilled water, and so on, until the eighth point, add 7 μl of 2ng / μl DNA and 3 μl trip...

Embodiment 3

[0025] Example 3 Extraction and Quantification of Serum DNA in Patients with Liver Cancer

[0026]Take 2ml of peripheral blood from 5 patients with liver cancer, let it stand at room temperature 20°C for 4 hours, centrifuge at 3000 rpm for 10 minutes, absorb the upper serum and then centrifuge to obtain a total of 700μl serum. Take 500 μl of serum, add 1000 μl of lysate DL and 11.5 μl of 20 mg / ml proteinase K (final concentration is 150 μg / ml), mix well and put it in a 65°C water bath for 15 minutes to 1 hour for digestion. The liquid is clear and not viscous. Take out the sample, add 1000 μl of isopropanol, mix it upside down, and then centrifuge at 12,000 rpm several times to make the liquid pass through the adsorption column that comes with the kit, and discard the centrifuged liquid. Add 500 μl of buffer W1 to the column, let it stand for 1 minute, and centrifuge at 12,000 rpm for 30 seconds to discard the liquid. Wash the column repeatedly with 500 μl of W1 solution, di...

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Abstract

The invention is a quantitative detecting technique of trace circular DNA based on fluorescent dye. It analyzes the strength of the fluorescence generated by combining the dye with purified DNA to determine the content of dissociative DNA in peripheral blood. It can accurately detect trace circular DNA down to a level of nano g, and has the characters of rapidness, simplicity and convenience, sensitivity, and accuracy. The determination of special people and tumor patient verifies that it has higher reliability, sensitivity and accuracy. It can be used in screening large-scale high-danger tumor people, early tumor diagnosis and dynamic observation of curative effect prognosis.

Description

technical field [0001] The invention belongs to the field of medical examination and relates to serological detection technology, in particular to a quantitative detection technology of trace circulating DNA based on fluorescent dye. Background technique [0002] Tumor is a disease that seriously endangers human health, and it has been the second leading cause of death in my country. Early diagnosis and treatment of tumors is the key to improving the cure rate. Recent studies have shown that during the growth of tumors, they can continuously release tumor cell DNA into the peripheral blood, so the circulating DNA content of patients can be higher than normal, and there are tumor-specific molecular genetic changes. Circulating DNA is cell-free DNA present in peripheral circulating serum / plasma. The level of circulating DNA can reflect the presence and severity of tumors, and is expected to become a new broad-spectrum tumor molecular marker. [0003] The content of circulat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/96
Inventor 高海峰屠红
Owner FUDAN UNIV
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