Microbial genome DNA (deoxyribonucleic acid) indirect extraction method for evaluating diversity of animal intestinal microflora

A technology of intestinal microorganisms and extraction methods, which is applied in the field of indirect extraction of microbial genome DNA, can solve the problems of low DNA recovery rate and difficult removal of pollutants, and achieve the effects of low cost, strong applicability, and simple operation

Inactive Publication Date: 2013-03-20
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a method for indirect extraction of microbial genome DNA for evaluating the diversity of animal intestinal microbial communities, using the average density of bacteria (1.1 μg / cm 3 ) is less than the average density of the stool sample (1.7-2.3 μg / cm 3 ), pre-treat the samples before cell lysis, and separate microbial cells from their intestinal samples to solve the problems of difficult removal of pollutants and low DNA recovery rate in the purification step after intestinal microbial cell lysis

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1) Add 0.5mL of pre-cooled sterile water to 0.5g of test mouse feces samples, mix well, vortex for 10 minutes, then add 0.5mL homogenization buffer A, vortex for 10 minutes, low speed (250g) Centrifuge at room temperature for 10 minutes, collect the supernatant and transfer to another centrifuge bottle.

[0037] 2) Add 1 mL of pre-cooled homogenization buffer B to the precipitate obtained in the previous step to wash and resuspend, vortex for 10 minutes, centrifuge at low speed (250g) for 10 minutes at room temperature, take the supernatant, and use the supernatant obtained in step 1) Combine and centrifuge at 10,000 rpm for 30 minutes at room temperature to recover bacterial cells, and discard the supernatant.

[0038] 3) Add 1.5ml of washing solution C to the bacterial cells obtained in the previous step, wash for 5 minutes, and centrifuge at 10,000 rpm for 30 minutes at room temperature to pellet the bacterial cells. Add 160ul lysozyme (50mg / ml) and 20ul proteinase K ...

Embodiment 2

[0049] 1) Add 0.5mL pre-cooled sterile water to 0.5g test rat feces sample, mix well, vortex for 10 minutes, then add 0.5mL homogenization buffer A, vortex for 10 minutes, low speed (250g) at room temperature Centrifuge for 10 minutes, collect the supernatant and transfer to another centrifuge bottle.

[0050] 2) Add 1 mL of pre-cooled homogenization buffer B to the precipitate obtained in the previous step to wash and resuspend, vortex for 10 minutes, centrifuge at low speed (250g) for 10 minutes at room temperature, take the supernatant, and use the supernatant obtained in step 1) Combine and centrifuge at 10,000 rpm for 30 minutes at room temperature to recover bacterial cells, and discard the supernatant.

[0051] 3) Add 1.5ml of washing solution C to the bacterial cells obtained in the previous step, wash for 5 minutes, and centrifuge at 10,000 rpm for 30 minutes at room temperature to pellet the bacterial cells. Add 160ul lysozyme (50mg / ml) and 20ul proteinase K (20mg / m...

Embodiment 3

[0062] 1) Add 0.5mL pre-cooled sterile water to 0.5g test white rabbit feces sample, mix well, vortex for 10 minutes, then add 0.5mL homogenization buffer A, vortex for 10 minutes, low speed (250g) at room temperature Centrifuge for 10 minutes, collect the supernatant and transfer to another centrifuge bottle.

[0063] 2) Add 1 mL of pre-cooled homogenization buffer B to the precipitate obtained in the previous step to wash and resuspend, vortex for 10 minutes, centrifuge at low speed (250g) for 10 minutes at room temperature, take the supernatant, and use the supernatant obtained in step 1) Combine and centrifuge at 10,000 rpm for 30 minutes at room temperature to recover bacterial cells, and discard the supernatant.

[0064] 3) Add 1.5ml of washing solution C to the bacterial cells obtained in the previous step, wash for 5 minutes, and centrifuge at 10,000 rpm for 30 minutes at room temperature to pellet the bacterial cells. Add 160ul lysozyme (50mg / ml) and 20ul proteinase ...

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Abstract

The invention relates to a microbial genome DNA (deoxyribonucleic acid) indirect extraction method for evaluating diversity of animal intestinal microflora. Before cell lysis, the method performs pretreatment on a sample and separates microbial cells from the excrement sample to avoid the problems that pollutants are difficult to remove and the DNA recovery rate is low in a purification step. According to the invention, phenol and chloroform are not used in the extraction process, thus harm to the physical health of experimenters is reduced. The OD260 / OD230 and OD260 / OD280 of the extracted intestinal microorganism DNA are approximate to standard values, and the intestinal microorganism DNA can be directly applied to molecular operation to evaluate the diversity of animal intestinal microflora.

Description

technical field [0001] The invention relates to an indirect extraction method of microbial genome DNA for evaluating the diversity of intestinal microbial communities of animals. Background technique [0002] There are a large number of microbial flora in the intestinal tract of animals, and these microorganisms have a huge impact on the nutrition, immunity, growth and development of the body. Their isolation and identification are very important for studying the function of intestinal flora and the relationship with the body. The traditional method is mainly to separate and cultivate intestinal microorganisms through selective medium, and then carry out the classification and identification of pure bacteria. This technique is not only time-consuming and labor-intensive, but can only detect cultivable microorganisms, but is powerless to a large number of unknown non-culturable microorganisms in the gut. With the rapid development of modern biotechnology in recent years, a ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 王金斌唐雪明李文祝子坪李鹏刘华白蓝蒋玮何建华陈大超赵凯
Owner SHANGHAI ACAD OF AGRI SCI
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