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Method and reagent for extraction of viral/bacterial nucleic acid in animal sample

A technology for animal samples and bacterial nucleic acids, applied in biochemical equipment and methods, microbial measurement/inspection, DNA preparation, etc., can solve problems such as inappropriate purification of viral genome nucleic acids, and achieve protection from degradation and inhibition of nuclease activity , fully dissolved effect

Active Publication Date: 2019-02-26
USTAR BIOTECHNOLOGIES (HANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this patent is that it uses a toxic phenol chloroform reagent and a high-speed centrifuge with a speed of more than 10,000g
Although the patent uses a 2,000g low-speed centrifuge, the extracted large nucleic acid fragments are mainly eukaryotic genomic nucleic acids from white blood cells, which are not suitable for purifying small fragments of viral genomic nucleic acids

Method used

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  • Method and reagent for extraction of viral/bacterial nucleic acid in animal sample
  • Method and reagent for extraction of viral/bacterial nucleic acid in animal sample
  • Method and reagent for extraction of viral/bacterial nucleic acid in animal sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] 1. Cleavage sample: Take 0.2ml of bovine serum sample to a 1.5ml centrifuge tube, add 0.2ml of lysate, shake and mix, and let it stand at room temperature for 15 minutes to obtain the lysis reaction solution;

[0059] The lysate contains 4 mol / L of guanidine thiocyanate, 7 mol / L of urea, 0.1 mol / L of sodium chloride, 20 mmol / L of disodium edetate, 20 mmol / L of trishydroxymethylaminomethane, and Tween-20 5wt%, sodium lauroyl sarcosinate 0.5%;

[0060] 2. Adsorption of nucleic acid: Add 0.24ml of ethanol to the lysis reaction solution, the volume concentration of ethanol is 95%, after fully mixing, transfer the liquid to the spin column, and place the spin column in a hand-held centrifuge. Centrifuge at 1000×g for 1 minute, discard the liquid in the collection tube, and put the spin column back into the collection tube;

[0061] 3. Cleaning: Add 0.5ml cleaning solution to the spin column, and place the spin column in the hand centrifuge. Centrifuge at 2000×g for 1 minut...

Embodiment 2

[0065] 1. Cleavage sample: Take 0.2ml of porcine plasma sample to a 1.5ml centrifuge tube, add 0.2ml of lysate, shake and mix, and let it stand at room temperature for 15 minutes to obtain the lysis reaction solution;

[0066] The lysate contains 3 mol / L of guanidine thiocyanate, 5 mol / L of urea, 0.05 mol / L of sodium chloride, 10 mmol / L of disodium edetate, 10 mmol / L of trishydroxymethylaminomethane, and Tween-20 3wt%, sodium lauroyl sarcosinate 0.3%;

[0067] 2. Adsorption of nucleic acid: Add 0.12ml of ethanol to the lysis reaction solution, the volume concentration of ethanol is 98%, after fully mixing, transfer the liquid to the spin column, and place the spin column in a hand-held centrifuge. Centrifuge at 500×g for 1 minute, discard the liquid in the collection tube, and put the spin column back into the collection tube;

[0068] 3. Cleaning: Add 0.5ml cleaning solution to the spin column, and place the spin column in the hand centrifuge. Centrifuge at 2000×g for 1 min...

Embodiment 3

[0072] 1. Cleavage sample: Take 0.3ml of bovine plasma sample to a 1.5ml centrifuge tube, add 0.3ml of lysate, shake and mix, and let it stand at room temperature for 10 minutes to obtain the lysis reaction solution;

[0073] The lysate contains guanidine thiocyanate 5mol / L, urea 8mol / L, sodium chloride 0.2mol / L, edetate disodium 30mmol / L, trishydroxymethylaminomethane 30mmol / L, Tween-20 6wt%, sodium lauroyl sarcosinate 0.8wt%;

[0074] 2. Adsorption of nucleic acid: Add 0.42ml of ethanol to the lysis reaction solution, the volume concentration of ethanol is 95%, after fully mixing, transfer the liquid to a spin column, and place the spin column in a hand-held centrifuge. Centrifuge at 2000×g for 1 minute, discard the liquid in the collection tube, and put the spin column back into the collection tube;

[0075] 3. Cleaning: Add 0.5ml cleaning solution to the spin column, and place the spin column in the hand centrifuge. Centrifuge at 2000×g for 1 minute, discard the liquid i...

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Abstract

The invention discloses a method and a reagent for extraction of viral / bacterial nucleic acid in an animal sample. The method comprises the following steps: taking the animal sample, and adding a lysate for lysis at a room temperature so as to obtain a lysis reaction solution; and adding an ethanol solution into the lysis reaction solution, transferring an obtained mixture into a silica gel containing adsorption membrane centrifugal column, carrying out adsorption with a manner of low-speed centrifugation, and carrying out washing and eluting. According to the invention, the bacterial nucleicacid extracted by using the method provided by the invention has high purity; no proteinase K for sample digestion is needed to be used in the process of extraction; reagent components and an operation flow are simplified; the cost of hardware equipment can be reduced through a manner of low-speed centrifugation in the process of extraction; a low-speed palm centrifuge can be adopted, is convenient to carry and is applicable to on-site operation; the components of the lysate are optimized; the binding ability of nucleic acid molecules to a silica-gel adsorption membrane is reinforced; and goodnucleic acid extraction efficiency is guaranteed.

Description

technical field [0001] The invention belongs to the field of nucleic acid extraction, and in particular relates to a method and reagent for extracting virus / bacterial nucleic acid from animal samples. Background technique [0002] Extracting nucleic acid from animal serum is an important experimental technique often used in molecular biology research. The extracted DNA and RNA templates can be used for molecular detection of related bacteria or viruses. [0003] Spin column method is one of the most commonly used nucleic acid extraction methods. This method utilizes the characteristic that nucleic acid molecules are bound to silica gel adsorption membrane in high-salt and low-pH environment, and separated from silica gel adsorption membrane in low-salt and high-pH environment. enrich and purify nucleic acid templates. Combined with a high-speed centrifuge or an automated nucleic acid extractor, the spin column method has the advantages of large throughput, fast speed, hig...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1006C12Q1/6806C12Q2523/308
Inventor 尤其敏朱勤锋朱罗罗袁旦一李玲饶焕新刘莹孙刚胡江文赵博熹胡林何谦
Owner USTAR BIOTECHNOLOGIES (HANGZHOU) CO LTD
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