A method for efficiently extracting intestinal contents and extracellular DNA of earthworms

Abstract

The invention discloses a method for efficiently extracting earthworm gut content and its extracellular DNA. The steps are as follows: rapid acquisition of earthworm gut content; construction and preparation of the extracellular genome internal standard gene; Extra DNA extraction. On the one hand, the method of the present invention can quickly take out the intestinal contents of individual tiny Eisenia chinensis, with simple steps and short time-consuming; on the other hand, it can also effectively separate and purify the extracellular eDNA in the intestinal contents, The extraction effect is stable, reliable and repeatable, and the extraction efficiency can reach 61%. The OD260 / OD280 of the extracted eDNA can reach 1.63, and the OD260 / OD230 can reach 1.72 through the detection of a nucleic acid protein analyzer, and subsequent molecular operations can be performed. The extraction method of the invention greatly improves the extraction quality of the intestinal content of the earthworm, and lays a foundation for the subsequent research on the extracellular genetic material of the earthworm intestinal tract.

Description

technical field [0001] The invention belongs to the field of biotechnology science and technology, in particular to a method for efficiently extracting earthworm intestinal contents and extracellular DNA. Background technique [0002] Earthworm transformation is an emerging technology for high-value and efficient resource utilization of livestock and poultry manure. This technology not only has high treatment efficiency, but also can turn waste into treasure and realize green environmental protection, which has attracted increasing attention. However, due to the long-term use of low-dose antibiotics in the breeding process, a large number of antibiotic-resistant bacteria (Antibiotic resistance bacteria, ARB) and antibiotic resistance genes (Antibiotic Resistance Genes, ARGs) have appeared in livestock and poultry manure, and the current pollution situation is very serious. severe. If left untreated, ARGs in livestock and poultry manure may be enriched in earthworms or remai...

AI Technical Summary

Problems solved by technology

[0004] At present, the intestinal contents of earthworms are mainly obtained by dissecting the earthworms, but this method is mainly suitable for the larger individuals of Trichos williams, and this method is time-consuming, and the intestinal contents are also easily polluted by body fluids However, there is still a lack of corresponding rapid extraction methods for the rapid acquisition of intestinal contents of individual smaller Eisenia chinensis; in addition, the extraction methods for eDNA in different samples mainly include phenol-chloroform extraction, magnetic bead adsorption method, CTAB extraction method, etc., but these methods are mainly aimed at the extraction of eDNA from tissue, plasma and cultured manure

However, the intestinal contents of earthworms are complex, and earthworm body fluids are seriously disturbed. The existing methods cannot be directly used for the extraction of eDNA in earthworm intestines. Therefore, it is very important to establish a method for rapid acquisition of earthworm intestinal contents and efficient extraction of eDNA.

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