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Reagent for extracting pathogenic nucleic acid from animal tissue sample

An animal tissue and reagent technology, applied in the biological field, can solve the problems of long time, cumbersome operation and lack, and achieve the effects of short time, high extraction efficiency and simple operation.

Active Publication Date: 2012-04-18
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purity of nucleic acid obtained by these classic methods is relatively high, which can basically meet the requirements of various tests such as subsequent PCR and enzyme digestion, but there are many defects: First, the entire operation process takes a long time, and the digested and broken cells need to be placed for 2-3 hours or overnight. The second is that the operation is cumbersome, and organic reagents such as phenol are required to be repeatedly extracted to remove proteins, and the Trizol method also requires low-temperature centrifugation at 4°C; A simple and fast nucleic acid extraction method for detection technology; Fourth, the extraction efficiency is limited and cannot meet the precise requirements of laboratory nucleic acid extraction

Method used

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  • Reagent for extracting pathogenic nucleic acid from animal tissue sample
  • Reagent for extracting pathogenic nucleic acid from animal tissue sample
  • Reagent for extracting pathogenic nucleic acid from animal tissue sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 extracts the preparation of pathogenic nucleic acid reagent

[0038] 1. Preparation of 0.75M sodium citrate (pH7.0): Weigh 11.03g sodium citrate, add 40mLddH 2 O, adjust the pH value to 7.0 with hydrochloric acid, and use ddH 2 O to a volume of 50 mL, autoclaved.

[0039] 2. Preparation of 10% SLS: Weigh 5g SLS into the reagent bottle, add 50mL ddH 2 O, heated and stirred to dissolve, filtered and sterilized to obtain the filtrate.

[0040] 3. Preparation of reagent for extracting pathogenic nucleic acid: Weigh 35.448g of guanidine isothiocyanate and put it into a sterile reagent bottle, add 1.667mL of 0.75M sodium citrate (pH7.0) solution, and 2.5mL of 10% SLS. Adjust the pH to 8.7 with NaOH, and dilute to 50 mL with sterile water. The final concentrations of each solute were: 6M guanidine isothiocyanate, 25mM sodium citrate, 0.5% SLS. Store at room temperature for later use.

Embodiment 2

[0041] Embodiment 2 extracts the preparation of pathogenic nucleic acid reagent

[0042] 1. The preparation of 0.75M sodium citrate (pH7.0) is the same as in Example 1.

[0043] 2. The preparation of 10% SLS is the same as in Example 1.

[0044] 3. Preparation of reagent for extracting pathogenic nucleic acid: Weigh 29.54 g of guanidine isothiocyanate into a reagent bottle, add 1.533 mL of 0.75M sodium citrate (pH7.00) solution, and 1.5 mL of 10% SLS. Adjust the pH to 7.0 with NaOH, and dilute to 50 mL with sterile water. The final concentrations of each solute were: 5M guanidine isothiocyanate, 23mM sodium citrate, 0.3% SLS. Store at room temperature for later use.

Embodiment 3

[0045] Embodiment 3 extracts the preparation of pathogenic nucleic acid reagent

[0046] 1. The preparation of 0.75M sodium citrate (pH7.0) is the same as in Example 1.

[0047] 2. The preparation of 10% SLS is the same as in Example 1.

[0048] 3. Preparation of reagent for extracting pathogenic nucleic acid: Weigh 23.632g of guanidine isothiocyanate into a reagent bottle, add 1.333mL of 0.75M (pH7.00) sodium citrate solution, and 1mL of 10% SLS. The pH was adjusted to 7.0 with NaOH, and the volume was adjusted to 50 mL with water. The final concentrations of each solute were: 4M guanidine isothiocyanate, 20mM sodium citrate, 0.2% SLS.

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Abstract

The invention provides a reagent for extracting pathogenic nucleic acid from an animal tissue sample. The pH of the reagent is 7 to 9, the solvent is water, and the solute contains the following substances with final concentration: 4 to 6 M of guanidinium isothiocyanate, 20 to 25 mM of sodium citrate, and 0.2 to 0.5 mass percent of sodium lauryl sulfate (SLS). The cost for extracting the nucleic acid by adopting the reagent is low; and the reagent is nontoxic, harmless, safe, quick and suitable for extracting the pathogenic nucleic acid in the animal tissue sample.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an extraction reagent for carrying pathogenic nucleic acid in animal tissue samples. Background technique [0002] In recent years, severe zoonotic diseases such as influenza A (H1N1) and avian influenza have exploded in the world, causing certain economic losses in China and threatening the health of Chinese people at all times; African swine fever, Rift Valley fever, etc. Animal diseases have approached the border, and new zoonotic diseases such as Nipah virus and Rift Valley fever have broken out in neighboring countries of our country. This series of potential animal diseases spreading across borders has posed a severe test to my country's prevention of high-risk animal diseases from spreading domestically. At the same time, with the development of my country's diplomatic and foreign trade undertakings, the number of imported and exported animals and animal products has increase...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/70C12Q1/68C12R1/93
Inventor 吴绍强林祥梅王彩霞邓俊花张丽张永宁
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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