A device and method for extracting the total DNA of substrate-attached microorganisms

Abstract

The invention discloses a device and method for extracting the total DNA of substrate-attached microorganisms, belonging to the technical field of molecular biology. The device includes a pressure-controlled flushing device, a negative pressure device and a collection device. The pressure of the flushing water is controlled by the pressure-controlled flushing device. The suspension produced by flushing the matrix through the negative pressure pipeline is collected to the collection device. The invention also discloses a DNA extraction method. The matrix is ​​washed under pressure, and the sample is collected with a negative pressure device, which overcomes the prior art. Aiming at the difficulty and incomplete sampling of microbial samples attached to the matrix, the representativeness, extraction efficiency and quality of the microbial genomic DNA attached to the matrix have been significantly improved, the bands are bright and clear, and there is basically no degradation. Fungi, etc. have good extraction efficiency.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a device and a method for extracting the total DNA of substrate-attached microorganisms. Background technique [0002] In recent years, with the development of sequencing technology, molecular biology methods for analyzing microbial diversity and function based on total deoxyribonucleic acid (DNA) in environmental samples have been continuously improved, including fluorescent quantitative nucleic acid amplification of a specific gene Detection (qPCR) method, metagenomic technology for sequencing the total DNA of microorganisms in the environment, etc., have gradually been widely used. Metagenomic technology is recognized as the most comprehensive and accurate method for studying environmental microbial community structure and functional genes, because it has no special selectivity (such as pure culture / enrichment), and it also reduces the preference of sequencing techno...

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Problems solved by technology

[0006] Although there have been various extraction methods developed for different researchers, these methods have their own defects in the DNA extraction of substrate-attached microorganisms in the environment, such as in-situ extraction, liquid nitrogen grinding, etc. The operation is time-consuming and labor-intensive, and multiple mortars need to be prepared. It is not easy to clean the equipment after grinding, which will easily contaminate the subsequent samples after extraction. Care must be taken during the extraction process. Do not shake to avoid DNA degradation; Bit extraction technology, one needs to completely immerse the small pieces of the matrix in the extraction solution, and the surface of the matrix must be destroyed, and the other is that the matrix itself is very easy to introduce impurities, and lysozyme is generally added to the extraction solution, causing the cell wall / membrane of microorganisms to rupture. After a large amount of biological macromolecules in the cells are dissolved, they will complex with impurities such as humic acid, extracellular proteins, polysaccharides, lipids, and heavy metals in the matrix, resulting in the DNA extracted by this method often requiring cumbersome post-processing steps. , resulting in a decrease in DNA extraction efficiency, and even a large amount of DNA degradation during the extraction process, resulting in the loss of DNA from low-abundance microorganisms below the detection limit, resulting in poor representativeness of the results; and in the ectopic extraction method, due to shaking The mechanical force is small, or because it is difficult for the cotton swab to penetrate into the pores of the porous substrate, the microorganisms that are often transferred are the outer layer of the biofilm, and it is difficult to shake or scrape the microorganisms that are tightly bound to the substrate from the substrate. extremely poor representation

There is not yet a general, fast and efficient method for obtaining representative and typical microorganisms from the attachment substrate and extracting DNA

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