Method for quickly extracting animal tissue virus nucleic acid based on nano magnetic beads and application

A technology for animal tissue and viral nucleic acid, which is applied in biochemical equipment and methods, microbial determination/inspection, recombinant DNA technology, etc., can solve the problems of nucleic acid extraction failure, interfere with PCR reaction, reduce yield, etc., and achieve simple and reliable operation. sexual effect

Inactive Publication Date: 2008-08-27
BEIJING JINNAXIN BIOTECH
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Problems solved by technology

[0004] However, there are some problems in the above method: 1) when the virus content in animal tissue is very low, the nucleic acid extraction often fails; 2) the purity of the extracted viral nucleic acid is not high, and contains a large amount of miscellaneous proteins that affect subsequent nucleic acid experiments, especially interference with PCR reactions; 3) operation steps Complicated and highly demanding for experimenters; 4) It takes a long time and nucleic acids, especially RNA, are easily degraded
Although the solid-phase adsorption nucleic acid method removes most of the foreign proteins, some nucleic acids are adsorbed on the surface of the solid-phase carrier to reduce the yield (see specific embodiment 1)

Method used

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  • Method for quickly extracting animal tissue virus nucleic acid based on nano magnetic beads and application

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specific Embodiment approach 1

[0019] Specific embodiment 1 Shellfish tissue virus nucleic acid extraction

[0020] Virus particle polyethylene glycol precipitation: Add 35ml of Gly-Tris buffer to 5g of hepatitis A virus-infected shellfish intestinal tissue and digestive gland tissue, fully homogenize in a homogenizer for 150s; centrifuge at 2000g for 5min, collect the supernatant; add polyethylene glycol Alcohol 4000 solution (8% final concentration) was mixed evenly, centrifuged at 10000 g for 10 min, and the supernatant was removed.

[0021] Extraction of nucleic acid by nano-magnetic beads method: 8nm, 600nm beads preparation methods refer to the relevant patent (application number 200710106001.4) or from Beijing Jinnaxin Biotechnology Co., Ltd.; add 50 μL of nano-magnetic beads and PBS buffer to the precipitation, mix well; put the centrifuge tube Place on a magnetic stand, absorb nano magnetic beads for 10 minutes, and remove the supernatant; wash the magnetic beads twice with deionized water and resu...

specific Embodiment approach 2

[0023] Specific embodiment 2 Extraction of virus nucleic acid from poultry tissue

[0024] Virus particle polyethylene glycol precipitation: Add 10ml of PBS buffer to 2g of chicken digestive tissue and muscle tissue infected with avian influenza virus, fully homogenize in a homogenizer for 150s; centrifuge at 5000g for 5min, collect supernatant; add polyethylene glycol 4000 solution (The final concentration is 10%) and mix well, centrifuge at 10000g for 10min, and remove the supernatant.

[0025] Extraction of Nucleic Acid by Nano-magnetic Beads: Same as Virus Extraction from Shellfish Tissue

[0026] Trizol-Chloroform Method for Extracting Nucleic Acids: Similar to Shellfish Tissue Virus Extraction Method

specific Embodiment approach 3

[0027] Specific embodiment 3 Fish tissue virus nucleic acid extraction

[0028] Virus particle polyethylene glycol precipitation: 2g of fish infected with hemorrhagic disease virus digested tissue was added to 10ml of PBS buffer, fully homogenized in a homogenizer for 150s; centrifuged at 5,000g for 5min, and the supernatant was collected; 10%), mix well, centrifuge at 10000g for 10min, and remove the supernatant.

[0029] Extraction of Nucleic Acid by Nano-magnetic Beads: Same as Virus Extraction from Shellfish Tissue

[0030] Trizol-Chloroform Method for Extracting Nucleic Acids: Similar to Shellfish Tissue Virus Extraction Method

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Abstract

The invention relates to a method, which comprises: homogenizing animal tissues, then, centrifugally taking the supernatant, centrifugally depositing virus particles with polyethylene glycol, evenly mixing sediment and nanometer magnetic beads to absorb the virus particles on the surface of the magnetic beads, heating and cracking virus to release nucleic acid, and thereby realizing the extraction of animal tissue viral nucleic acid. The method can extract micro viral nucleic acid and can effectively remove PCR inhibitory protein with simple and reliable operation.

Description

[technical field] [0001] The invention relates to the field of isolation and identification of virus nucleic acid from animal tissues. [Background technique] [0002] Molecular biological identification of virus in animal tissue is the basis for studying molecular pathology and preventing and controlling virus transmission. The extraction of viral nucleic acid is a prerequisite for molecular biological identification. The composition of animal tissues is complex and the amount of virus is small, which makes the yield and purity of nucleic acid very low. Nucleic acid products contain a large number of foreign proteins, which seriously interfere with subsequent experiments. [0003] The nucleic acid extraction process is roughly divided into three parts: 1) virus release; 2) virus enrichment; 3) virus lysis and nucleic acid purification. In order for the virus to be released from the tissue, the tissue first needs to be homogenized. After homogenization, there are floccule...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/70C12Q1/68
Inventor 聂棱江明
Owner BEIJING JINNAXIN BIOTECH
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