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Reagent for extracting RNA or DNA virus in body fluid

A technology of reagents and body fluids, applied in the field of biochemistry, can solve the problems of cumbersome steps, low efficiency, and low extraction efficiency, and achieve the effects of reducing protein impurities, reducing co-precipitation, and improving utilization

Inactive Publication Date: 2009-10-28
GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The mainstream method for extracting viral RNA / DNA in plasma and serum is the "Trizol" method, but this method needs to absorb the supernatant and change the tube during the extraction process. The supernatant cannot be completely aspirated, and some RNA / DNA will always be lost. More cumbersome, so not very suitable for strict absolute quantification
In addition, one-tube viral RNA / DNA extraction reagents have also appeared on the domestic and foreign markets, but the extraction efficiency of this type of reagent is low, and a liquid sample of more than 200ul is required to achieve high sensitivity, such as the HIV quantitative reagent of Roche And domestic Tianze gene RNAout extraction reagent
Roche’s HIV quantitative reagent can make the HIV quantitative sensitivity reach 200copies / ml with 200ul plasma, and the RNAout extraction reagent can make the virus quantitative sensitivity reach 50-100copies / ml with 200ul plasma according to its advertisement, but in fact due to the reagent A large amount of protein is precipitated while viral RNA is precipitated, and the final RNA dissolution volume needs to be more than 100ul, so its efficiency is not high, thus hindering its large-scale application

Method used

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  • Reagent for extracting RNA or DNA virus in body fluid
  • Reagent for extracting RNA or DNA virus in body fluid
  • Reagent for extracting RNA or DNA virus in body fluid

Examples

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example 1

[0021] The reagent composition of the invention: 5.4 mol guanidine isothiocyanate, 2 mol guanidine hydrochloride, 25 mmol sodium citrate, 20 ml β-mercaptoethanol, 7 g sodium lauryl sarcosinate, 2 mol urea, 64 mg glycogen, and the rest is water.

[0022] The preparation of the reagent of the present invention: take guanidine isothiocyanate, guanidine hydrochloride, sodium citrate 2, β-mercaptoethanol, sodium lauryl sarcosinate, urea and glycogen and mix them with DEPC-treated water to dissolve and Quantify to 1L, then filter with 0.45μm membrane.

[0023] Extraction of viral RNA:

[0024] (1) Material: Dilute the SIV virus standard (international standard SIV virus particle standard, gifted by Professor Lu Wei, University of Paris Fifth, France) with monkey plasma to 6 concentrations, respectively: 0.5×10 7 copies / ml, 0.5×10 6 copies / ml, 0.5×10 5 copies / ml, 0.5×10 4 copies / ml, 0.5×10 3 copies / ml, 0.5×10 2 copies / ml, 12 parallel extraction experiments for each concentration. At the ...

example 2

[0052] The reagent composition of the present invention is 5.4 mol of guanidine isothiocyanate, 2 mol of guanidine hydrochloride, 25 mmol of sodium citrate, 20 ml of β-mercaptoethanol, 7 g of sodium lauryl sarcosinate, 2 mol of urea, 64 mg of glycogen, and the rest is water.

[0053] The preparation of the reagent of the present invention: Take guanidine isothiocyanate, guanidine hydrochloride, sodium citrate, β-mercaptoethanol, sodium lauryl sarcosinate, urea and glycogen and mix them with DEPC-treated water to dissolve and quantify To 1L, then filter with 0.45μm filter membrane.

[0054] Experimental materials: serum samples of patients with HBVDNA quantitative copy number above 1e9 tested in the hospital.

[0055] Dilute HBVDNA patient serum samples with cell culture newborn calf serum, and dilute to: 5e8 copies / ml, 5e7 copies / ml, 5e6 copies / ml, 5e5 copies / ml, 5e4 copies / ml, 5e3 copies / ml, 5e2 copies / Ml, 5e1 copies / ml, 3 replicate holes for each concentration, and 1 hole of ne...

example 3

[0069] The reagent composition of the present invention: 4mol of guanidine isothiocyanate, 1mol of guanidine hydrochloride, 20mmol of sodium citrate, 10ml of β-mercaptoethanol, 5g of sodium lauryl sarcosinate, 1.5mol of urea, 60mg of glycogen, and water as the balance.

[0070] The preparation of the reagent of the present invention: take guanidine isothiocyanate, guanidine hydrochloride, sodium citrate, β-mercaptoethanol, sodium lauryl sarcosinate, urea and glycogen, and mix them with DEPC-treated water to dissolve and quantify To 1L, then filter with 0.45μm filter membrane.

[0071] Experimental materials: serum samples of patients with HBVDNA quantitative copy number above 1e9 tested in the hospital.

[0072] Dilute HBVDNA patient serum samples with cell culture newborn calf serum, and dilute to: 5e8 copies / ml, 5e7 copies / ml, 5e6 copies / ml, 5e5 copies / ml, 5e4 copies / ml, 5e3 copies / ml, 5e2 copies / Ml, 5e1 copies / ml, 3 replicate holes for each concentration, and 1 hole of newborn...

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Abstract

The invention provides a reagent for extracting RNA or DNA virus in body fluid, which is characterized in that each liter of the reagent comprises the following compositions: 4 to 6 mols of guanidinium isothiocyanate, 1 to 2.5 mols of guanidine hydrochloride, 20 to 50 mmols of sodium citrate, 10 to 30 ml of beta-mercaptoethanol, 5 to 10 grams of sarcosyl, 1.5 to 3 mols of urea, 60 to 100 mg of glycogen, and the balance of water. The efficiency of extracting the RNA or DNA virus by the reagent is high, and high-purity RNA or DNA virus can be stably obtained only by 100 mu L of body fluid sample. When the obtained RNA or DNA is used for quantitative PCR detection, the detection sensitivity can reach 50 copies / mL.

Description

Technical field [0001] The present invention relates to the field of biochemistry, in particular to the separation of nucleic acids. Background technique [0002] The mainstream method for extracting viral RNA / DNA in plasma and serum is the "Trizol" method, but this method requires the supernatant to be sucked and the tube replaced during the extraction process. The supernatant cannot be completely aspirated, and part of the RNA / DNA is always lost. It is more complicated, so it is not very suitable for strict absolute quantification. In addition, a tube of viral RNA / DNA extraction reagents has appeared on the market in my country and abroad, but the extraction efficiency of these reagents is low, and more than 200ul of liquid samples are required to achieve high sensitivity, such as Roche's HIV quantitative reagents. And domestic Tianze gene's RNAout extraction reagent. Roche's HIV quantitative reagent uses 200ul plasma to make HIV quantitative sensitivity reach 200copies / ml, whi...

Claims

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Application Information

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IPC IPC(8): C12N15/10C07H21/00
Inventor 何金洋
Owner GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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