Method of simultaneously extracting animal DNA (Deoxyribonucleic Acid) virus and RNA (Ribonucleic Acid) virus nucleic acid in blood serum and double swabs

A DNA virus, RNA virus technology, applied in DNA preparation, recombinant DNA technology and other directions, can solve the problems of high cost, low extraction efficiency, and cumbersome extraction process.

Inactive Publication Date: 2014-03-05
安徽华卫集团禽业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The classic Trizol extraction method is cumbersome, long cycle, low extraction efficiency, and high technical requirements. The silica gel mold col...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0022] Preparation and use of liquid:

[0023] Guanidine isothiocyanate lysate: 5M guanidine isothiocyanate, 0.8% TriTon-100, 40mM Tris-Cl, 10mM DTT, 80μg / mL proteinase K, 20mM EDTA, pH 4.5.

[0024] Lotion Ⅰ: The composition is 5.5M guanidine hydrochloride, 56% absolute ethanol, 80 μg / mL proteinase K, and the pH is 6.5.

[0025] Lotion II: The composition is 75% ethanol.

Embodiment example 2

[0027] Simultaneously extract the method for animal DNA virus and RNA virus nucleic acid in serum, double swab, comprise steps:

[0028] (1) Take a clean centrifuge tube, add 100 μL sample to 200 μL guanidine isothiocyanate lysate, vortex and mix, and let stand for 5 minutes;

[0029] (2) Add the same volume of absolute ethanol as the guanidine isothiocyanate lysate, that is, 200 μL, and invert and mix repeatedly for 15 seconds;

[0030] (3) Put the filter column on the 2mL collection tube, transfer the whole mixture to the filter column, and centrifuge at 12,000rpm for 1 minute;

[0031] (4) Discard the liquid in the collection tube, add 600 μL of wash solution 1 to the filter column, and centrifuge at 12,000 rpm for 1 minute;

[0032] (5) Discard the liquid in the collection tube, add 600 μL of wash solution 2 to the filter column, and centrifuge at 12,000 rpm for 1 minute;

[0033] (6) Repeat step (5) once;

[0034] (7) Discard the liquid in the collection tube, put the ...

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PUM

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Abstract

The invention discloses a method of simultaneously extracting animal DNA (Deoxyribonucleic Acid) virus and RNA (Ribonucleic Acid) virus nucleic acid in blood serum and double swabs. The method comprises the following steps of: carrying out lysis on a to-be-extracted substance by using guanidinium isothiocyanate lysate; adsorbing RNA by a silica gel membrane; removing impure protein by washing liquor I; removing impurities by washing liquor II; carrying out DEPC (Diethylpyrocarbonate) water-washing to remove nucleic acid, wherein the guanidinium isothiocyanate lysate comprises 3M-7M guanidinium isothiocyanate, 0.6%-1.0% TriTon-100, 30mM-50mM Tris-Cl, 5mM-15mM DTT (DL-Dithiothreitol), 60 mu g/mL-90 mu g/mL protease K, 10mM-30mM EDTA (Ethylene Diamine Tetraacetic Acid), and PH of the guanidinium isothiocyanate lysate is 4.3-4.6; the washing liquor I comprises 5M-6M guanidine hydrochloride, 53%-59% absolute ethyl alcohol, 70-90 mu g/mL protease K, and PH of the washing liquor I is 6.4-6.6; the washing liquor II comprises 70%-80% alcohol. The method disclosed by the invention has the advantages of being simple in extracting process, short in period, low in cost, and capable of simultaneously extracting RNA virus and DNA virus nucleic acid in an animal blood serum sample and a double-swab sample.

Description

technical field [0001] The invention relates to a method for simultaneously extracting animal DNA virus and RNA virus nucleic acid from serum and double swabs, which is suitable for the field of biotechnology. Background technique [0002] With the rapid development of molecular biology, genetic diagnosis has become the most accurate and effective means of detecting animal diseases. In the process of genetic diagnosis, the extraction of viral nucleic acid is a prerequisite for detection, and the quality of nucleic acid extraction also affects the accuracy of detection. . Currently, animal virus nucleic acid extraction methods mainly include the classic Trizol extraction method and the silica gel molded column extraction method using Trizol as the lysate. The classic Trizol extraction method is cumbersome, long cycle, low extraction efficiency, and high technical requirements. The silica gel mold column extraction method using Trizol as the lysate is relatively expensive, th...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 赵宏波陈帮照
Owner 安徽华卫集团禽业有限公司
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