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Reagent for extracting RNA or DNA virus in body fluid

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

Inactive Publication Date: 2011-08-10
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 present invention: 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 sarcosine, 2 mol of urea, 64 mg of glycogen, and the rest is water.

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

[0023] Extraction of viral RNA:

[0024] (1) Materials: Dilute the SIV virus standard product (international general SIV virus particle standard product, gifted by Professor Lu Wei of Paris Fifth University, 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 ...

example 2

[0050] The reagent composition of the present invention: 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 sarcosine, 2 mol of urea, 64 mg of glycogen, and the rest is water.

[0051] Preparation of reagent of the present invention: get guanidine isothiocyanate, guanidine hydrochloride, sodium citrate, β-mercaptoethanol, sodium lauryl sarcosinate, urea and glycogen mix, dissolve and quantify with DEPC-treated water to 1 L, and then filtered through a 0.45 μm filter.

[0052] Experimental materials: Serum samples from patients with a quantitative HBVDNA copy number above 1e9 detected clinically in the hospital.

[0053] Dilute the serum samples of HBVDNA patients with newborn bovine serum for cell culture, and the dilutions are: 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 duplicate holes are set for each...

example 3

[0066] The reagent composition of the present invention: 4 mol of guanidine isothiocyanate, 1 mol of guanidine hydrochloride, 20 mmol of sodium citrate, 10 ml of β-mercaptoethanol, 5 g of sodium lauryl sarcosine, 1.5 mol of urea, 60 mg of glycogen, and the balance is water.

[0067] Preparation of reagent of the present invention: get guanidine isothiocyanate, guanidine hydrochloride, sodium citrate, β-mercaptoethanol, sodium lauryl sarcosinate, urea and glycogen mix, dissolve and quantify with DEPC-treated water to 1 L, and then filtered through a 0.45 μm filter.

[0068] Experimental materials: Serum samples from patients with a quantitative HBVDNA copy number above 1e9 detected clinically in the hospital.

[0069] Dilute the serum samples of HBVDNA patients with newborn bovine serum for cell culture, and the dilutions are: 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 duplicate holes are set for e...

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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 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 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. Relatively cumbersome, so it is not very suitable for strict absolute quantification. In addition, one-tube virus RNA / DNA extraction reagents have also appeared in my country and foreign markets, but the extraction efficiency of such reagents is low, and liquid samples above 200ul are required to achieve high sensitivity, such as the HIV quantitative reagent of Roche And domestic Tianze gene RNAout extraction reagents. Roche’s HIV quantitative reagent can make the HIV quantitative sensitivity reach 200copies / ml with 200ul plasma, and the RNAout ex...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C07H21/00
Inventor 何金洋
Owner GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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