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DNA extracting method for evaluating community diversity of the intestinal microorganisms of animals

A technology for intestinal microorganisms and extraction methods, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA preparation, etc., can solve the problems of low DNA recovery rate, difficult removal of pollutants in purification steps, etc. Reduce physical health damage, strong applicability effect

Inactive Publication Date: 2010-05-26
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 DNA extraction method for evaluating the diversity of animal intestinal microbial communities, so as 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

[0034] (1) Add 1.5 ml of sample soaking solution A to 0.5 g of mouse feces sample, vortex for 10 minutes, centrifuge at 10,000 rpm for 5 minutes, discard the supernatant, and repeat 3 times.

[0035] (2) Add 1.5ml of sample immersion solution 2, place on ice for 5 minutes, vortex for 10 minutes, centrifuge at 10,000 rpm for 5 minutes, discard the supernatant, and repeat 3 times.

[0036] (3) Add 0.3 g of quartz sand and 1 glass bead with a diameter of 4 mm, and add 978 μl of phosphate buffer C (pH=8.0) to the sample. Vortex for 5 minutes.

[0037] (4) Add 122 μl of cell lysate D to the sample, and vortex for 5-20 minutes. Centrifuge at 13000rmp for 10min to precipitate debris.

[0038] (5) Transfer the supernatant to a clean centrifuge tube, add 250 μl of protein removal solution E to the tube, gently invert and mix 10 times by hand, leave at room temperature for 5 minutes, and centrifuge at 13,000 rpm for 5 minutes.

[0039] (6) Transfer the supernatant to a clean centrifu...

Embodiment 2

[0048] (1) Add 1.5ml sample soaking solution A to 0.5g rat feces sample, vortex for 10 minutes, 10000 rpm, centrifuge for 5 minutes, discard the supernatant, repeat 3 times.

[0049] (2) Add 1.5ml sample immersion solution 2, place on ice for 5 minutes, vortex for 10 minutes, 10000 rpm, centrifuge for 5 minutes, discard the supernatant, repeat 3 times.

[0050] (3) Add 0.3 g of quartz sand and 1 glass bead with a diameter of 4 mm, and add 978 μl of phosphate buffer C (pH=8.0) to the sample. Vortex for 5 minutes.

[0051] (4) Add 122 μl of cell lysate D to the sample, and vortex for 5-20 minutes. Centrifuge at 13000rmp for 10min to precipitate debris.

[0052] (5) Transfer the supernatant to a clean centrifuge tube, add 250 μl of protein removal solution E to the tube, gently invert and mix 10 times by hand, leave at room temperature for 5 minutes, and centrifuge at 13,000 rpm for 5 minutes.

[0053] (6) Transfer the supernatant to a clean centrifuge tube, add 3 times the vo...

Embodiment 3

[0062] (1) Add 1.5ml sample soaking solution A to 0.5g rabbit feces sample, vortex for 10 minutes, 10000 rpm, centrifuge for 5 minutes, discard the supernatant, repeat 3 times.

[0063] (2) Add 1.5ml sample immersion solution 2, place on ice for 5 minutes, vortex for 10 minutes, 10000 rpm, centrifuge for 5 minutes, discard the supernatant, repeat 3 times.

[0064] (3) Add 0.3 g of quartz sand and 1 glass bead with a diameter of 4 mm, and add 978 μl of phosphate buffer C (pH=8.0) into the sample. Vortex for 5 minutes.

[0065] (4) Add 122 μl of cell lysate D to the sample, and vortex for 5-20 minutes. Centrifuge at 13000rmp for 10min to precipitate debris.

[0066] (5) Transfer the supernatant to a clean centrifuge tube, add 250 μl of protein removal solution E to the tube, gently invert and mix 10 times by hand, leave at room temperature for 5 minutes, and centrifuge at 13,000 rpm for 5 minutes.

[0067] (6) Transfer the supernatant to a clean centrifuge tube, add 3 times t...

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Abstract

The invention discloses a DNA extracting method for evaluating the community diversity of the intestinal microorganisms of animals. Before cell disruption, a sample is pretreated, and DNA and pollutants outside the cells are removed, thereby avoiding the problems of difficult removal of the pollutants and low recovery rate of the DNA in a purification step. In the extracting process of the invention, phenol and chloroform are not used, and damage to the health of experiment personnel is reduced; and the obtained DNA is integral and has high yield, and the molecular fragment is larger than 10kb. The OD260 / OD230 and OD260 / OD280 of the DNA of the extracted intestinal microorganisms are close to standard values and can be directly applied to molecule operation to evaluate the community diversity of the intestinal microorganisms of animals.

Description

technical field [0001] The invention relates to a DNA extraction method for evaluating the diversity of intestinal microbial communities in 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 large number of molecular biol...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 唐雪明王金斌赵凯谭芙蓉吴潇朱宏陶世如蒋玲曦王利刚刘华
Owner SHANGHAI ACAD OF AGRI SCI
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