Process for abstracting total DNA of swine waste sample

An extraction method and feces sample technology, applied in the field of microbiology and molecular biology, can solve the problems of complex composition of animal feces, difficulty in selecting glass filters, and difficulty in popularization

Inactive Publication Date: 2008-11-19
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Such as Walsh etc. (referring to: Walsh P S, Metzger DA, Higuchi R.Bio Techniques, 1991,10 (4): 506-513) utilize Chelex-100 boiling method to successfully extract human DNA, this method is simple and easy, and it also The ability of Chelex-100 basic polyvalent metal ion chelating agent to combine with various metal ions (including metal ions necessary to maintain DNase activity) can be used to effectively inhibit the activity of DNase and prevent further degradation of DNA; but in The extraction process will lead to the denaturation of DNA molecules; and although the widely used magnetic bead adsorption method does not need to use protease or organic extraction reagents, it can directly use magnetic beads to absorb DNA, but when DNA is eluted from magnetic beads, it will be A large amount of DNA is lost, resulting in relatively low DNA yield (cf.: Flagstad O, Roed K, Stacy JE, et al. Mol Ecol, 1999, 8(5):879-883)
In addition, Yang Dejun et al. (see: Yang Dejun, Wu Jin, Liu Yi, etc. A method for rapidly extracting the total DNA of intestinal mi

Method used

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  • Process for abstracting total DNA of swine waste sample
  • Process for abstracting total DNA of swine waste sample
  • Process for abstracting total DNA of swine waste sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: The method of the present invention extracts the total DNA of a swine feces sample.

[0036] The samples used in the experiment were the collected feces of Rongchang pigs, and the collected feces samples were stored at -20°C for future use. Specific steps are as follows:

[0037] (1) Weigh an appropriate amount of stool sample into a sterile 50ml centrifuge tube A, add 10-20ml PBS buffer solution and sterile glass beads and vortex to mix;

[0038] (2) After centrifuging at 200-400xg for 2-8 minutes, take out as much supernatant suspension as possible into another sterile 50ml centrifuge tube B;

[0039] (3) Wash the precipitate obtained in step (2) again with an appropriate amount of PBS buffer, centrifuge at 200-400xg for 2-8min, take out the upper layer suspension and combine it into centrifuge tube B;

[0040] (4) Add an appropriate volume of paraformaldehyde solution of a certain concentration to the centrifuge tube B, and incubate it at 4°C for 1-2 ...

Embodiment 2

[0053] Embodiment 2: The comparison between the present invention and the conventional method and kit method for extracting total DNA from feces.

[0054] In order to compare the parallel effect of the present invention with the existing conventional method and the commercial kit extraction method, the applicant carried out a comparison experiment using the method of the present invention, the conventional method and the kit method to extract at the same time, using the conventional method to extract total DNA from feces : According to the method reported by Rousselon et al. (see: Rousselon N, Delgenès JP, Godon JJ.J Microbiol Methods, 2004, 59(1): 15-22). Utilize kit method to extract total DNA from feces: the kit used is purchased from QiaGen company, and the commodity name of kit is DNA Stool Mini Kit. The feces samples used in the above two methods are the same as the present invention, and the extracted DNA is dissolved in 100 ml of TE buffer.

[0055] Adopt the method...

Embodiment 3

[0056] Example 3: The DNA extracted in Example 2 was subjected to PCR amplification detection of 16S rDNA.

[0057] Using the DNA extracted in Example 2 as a template, with the designed primers: the forward primer P0 is 5'-GAGAGTTTGATCCTGGCTCAG-3' and the reverse primer 1492r is 5'-CGGCTTACCTTGTTACGACTT-3' to amplify the 16S rDNA (16S ribosomal RNA gene). Utilize 1 μl of feces total DNA prepared by the conventional method, the kit method and the method of the present invention as a template for PCR reaction to amplify the 16S rDNA of fecal microorganisms respectively. The PCR reaction system is as follows (20 μl): 10×PCR buffer, 2 μl; 2.0 mMMgCl2; 200 μM dNTP; 0.5 μM primer; 1U Taq enzyme (TaKaRa); DNA template, 1 μl; add ddH20 to 20 μl; ℃ 5min; denaturation: 95℃ 30s; annealing: 58℃ 30s; extension: 72℃ 1.5min; final extension 72℃ 8min; 30 cycles.

[0058] After amplification, take 8 μl and perform electrophoresis detection on a 1% agarose gel. The size of the amplified targe...

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Abstract

The invention relates to the microbiology and molecular biology technical field, in particular to a method for extracting pig manure sample total DNA. The method mainly comprises manure sample pretreatment and sample total DNA extraction. Moreover, the method comprises the following steps that: collected fresh manure sample or frozen manure sample is taken as a material; the pretreatment of the manure sample is carried out by means of a PBS buffer solution, a paraformaldehyde solution and anhydrous alcohol so as to obtain manure sample bacteria suspension; and a CTAB solution and beta-mercaptoethanol, etc. are used to carry out cell fragmentation, thereby releasing DNA. The method has the characteristics of simple operation, quickness, high efficiency and ideal repeatability and economical efficiency, etc.; moreover, the extracted DNA has high concentration, purity and yield, and can be directly used in subsequent molecular biology experimental researches such as PCR amplification without purification.

Description

[Field of invention] [0001] The invention belongs to the technical field of microbiology and molecular biology, and in particular relates to a method for extracting total DNA from pig feces samples. [Background technique] [0002] There are about 30 genera and more than 500 different bacteria in the intestinal tract of mammals, about 1×10 14 Living bacteria form a complex and dynamically balanced micro-ecological system, which plays a very important role in the digestion and absorption of host nutrients, the activation of immune mechanisms, and growth and development. If this micro-ecological balance is out of balance, the normal physiological functions of the animal body will be disturbed, leading to the occurrence and prevalence of diseases, which poses a great potential threat to animal husbandry, especially large-scale breeding. Therefore, in recent years, the research on the intestinal microflora structure and its diversity has become a current hot spot. However, due t...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 王红宁唐俊妮曾志光樊汶樵谢波曾瑜虹杨鑫
Owner SICHUAN UNIV
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