Extraction method of fly intestinal microorganism total DNA (deoxyribonucleic acid)

Abstract

An extraction method of the fly intestinal microorganism total DNA (deoxyribonucleic acid) relates to the technical field of experiment of molecular biology. The phenol-chloroform method is the traditional method for extracting bacterial DNAs, but the method is generally used in the DNA extraction of pure bacteria cultured in the laboratory. As the matrix ingredients and microorganisms of the environment sample are more complicated, the phenol-chloroform method and the kit method have the defects such as low cell lysis rate and large DNA loss when used for extracting total DNA; and the residual enzyme inhibitor in the extract can affect the subsequent polymerase chain reaction (PCR) amplification effect and the enzymatic reaction effect. Therefore, the extraction method is improved in the aspects such as the sample pretreatment, the operating step and the reagent solution; and in the method, the sodium-tris-EDTA (STE) buffer is used to pretreat the sample and lysozyme is used to ensure the completely splitting of Gram-positive cocci. When the fly intestinal microorganism total DNA extracted by the method is used for PCR amplification and 16S rRNA (ribosomal ribonucleic acid) library construction, the effect is better than that of domestic and import kits. The operating steps of the method are simple, and the pollution of the operating process to the sample and the damage of the operating process to DNA molecules can be effectively reduced.

Description

technical field [0001] The invention relates to molecular biology laboratory technology, in particular to an improved method for extracting the total DNA of intestinal microbes of flies. Background technique [0002] Fly is an insect that is closely related to human life. Due to its unique living habits, it is related to the transmission of many animal and human diseases, and has been identified as a vector by the World Health Organization. Because flies have lived in areas with poor sanitation for a long time, there are unique microbial communities in their bodies. These microbial communities not only play an important role in the nutritional development of flies, but also a large number of pathogenic bacteria in them continue to stimulate the natural immunity of flies. system, producing antimicrobial peptides. Therefore, the study of the intestinal microbial community of flies can not only discover new microbial species and reveal the law of disease transmission, but also...

AI Technical Summary

Problems solved by technology

However, the composition of samples from fly intestines is very complex, especially organic substances such as lipids and acids are difficult to remove during the total DNA extraction process, which directly affects the effect of subsequent PCR amplification and enzyme digestion reactions

In addition, after a small amount of fly intestinal samples undergo multiple steps such as cell lysis and DNA purification, the yield is greatly reduced, and the final total DNA concentration often cannot meet the needs of the next molecular operation.

Most current total DNA extraction methods have drawbacks, such as incomplete cell lysis, enzyme inhibitors in DNA extracts, and DNA loss, degradation, and shearing issues

[0005] Therefore, in order to solve the problems of enzyme reaction inhibitors, low cell lysis rate, and serious DNA loss in DNA samples obtained by conventional methods, it is necessary to study a method suitable for the extraction of total DNA from fly intestinal microorganisms.

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