Process for abstracting total DNA of swine waste sample

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...

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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 microorganisms, Chinese Journal of Microecology, 2006, 18(2): 91-93) used glass filters and reagents The DNA in the rabbit feces sample is extracted by combining the cassette method. The DNA extracted by this method is of high purity. However, due to the complex composition of animal feces, it is very difficult to select a glass filter with a suitable pore size for different animal feces. Therefore, , this method is difficult to popularize, and the extraction cost is high due to the use of kits, so it is not suitable for large-scale experimental extraction

[0006] To sum up, the conventional method and its improved method require further purification to obtain total DNA from feces before it can be used in subsequent experimental operations, which takes a long time and is not conducive to large-scale experimental extraction.

Although the new methods developed in recent years can overcome the shortcomings of conventional methods and improve the purity of the obtained DNA, they have brought problems such as DNA denaturation and cumbersome operations.

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