CN-122012242-A - Method for enriching fungi from ruminant gastrointestinal tract content and application thereof

Abstract

The invention discloses a method for enriching fungi from gastrointestinal tract contents of ruminants and application thereof, and relates to the technical field of microbial separation, wherein the method comprises screening and confirming a target sampling part, preprocessing and physical screening of abomasum contents, compound enzyme digestion treatment, sucrose density gradient centrifugation enrichment of fungi cells, deep purification of the fungi cells and verification of enrichment effect; according to the method, firstly, the abomasum is determined to be the most abundant target part of fungi through fungus specificity qPCR analysis, then physical screening and vacuum suction filtration are carried out on the abomasum content, then compound enzyme liquid is adopted for digestion, the combination of the fungi and plant matrixes is dissociated, background DNA is degraded, then fungus cells enriched in 30%, 45% and 60% concentration layers are selectively collected by utilizing a sucrose density gradient centrifugation technology, and finally, the detection sensitivity and the specificity of fungus ITS genes can be obviously improved through PCR and qPCR verification.

Inventors

• LI CUNYUAN
• LI XIAOYUE
• HAN LIN
• HU SHENGWEI
• HE HUINING
• YU JINMING

Assignees

• 石河子大学

Dates

Publication Date

20260512

Application Date

20260226

Claims (9)

1. 1. A method of enriching fungi from ruminant gastrointestinal contents, comprising the steps of: collecting fresh contents of different parts of the gastrointestinal tract of ruminants, and screening out a target part with highest fungus abundance through fungus specificity qPCR analysis; the content of the screened target part is physically screened, and then the large-particle impurities are removed by vacuum filtration, and the sediment is collected by centrifugation; Step three, re-suspending the centrifugally collected precipitate in a complex enzyme digestion solution for overnight digestion; step four, subjecting the sample after overnight digestion to sucrose density gradient centrifugation, and collecting fungal cells of a target concentration layer; Re-suspending the collected fungus cell precipitate in PBS buffer solution containing lysozyme for overnight digestion, adding DNaseI for further digestion for 1h, centrifuging and collecting the precipitate to finish deep purification; and step six, verifying the enrichment effect of the collected fungal cells.
2. 2. A method of enriching fungi from the contents of the gastrointestinal tract of a ruminant animal according to claim 1, wherein in step one, the ruminant animal is a dairy cow and the different parts of the gastrointestinal tract include the reticulum, rectum, colon, cecum, abomasum, ileum, valve stomach, small intestine and rumen.
3. 3. The method of claim 1, wherein in the second step, the physical sieving is performed by suspending and stirring the target part of the content with PBS buffer solution, sequentially filtering with different pore size screens from large to small, and sequentially performing vacuum filtration with 150 μm and 100 μm filter membranes.
4. 4. The method of claim 1, wherein in step three, the complex enzyme digest comprises cellulase, chitinase, pectase, glucanase, lysozyme, muramidase and DNaseI.
5. 5. A method for enriching fungi from ruminant gastrointestinal contents according to claim 1, wherein in step four, a discontinuous density gradient is constructed from bottom to top by using 60%, 45%, 30% and 15% sucrose solutions when sucrose density gradient centrifugation is performed, and after overnight digestion, the sample is added to the top layer of the gradient solution and centrifuged at 12000rpm for 10min, wherein the target concentration layer is 30%, 45% and 60% sucrose concentration layer, and 15% sucrose concentration layer is discarded.
6. 6. The method of claim 1, wherein in the fifth step, the collected precipitate is centrifuged at 3000rpm for 5min, and the collected precipitate is frozen in liquid nitrogen and then stored at-80 ℃.
7. 7. The method for enriching fungi from the gastrointestinal contents of ruminants according to claim 1, wherein in the step six, the specific steps of performing enrichment effect verification are that the DNA of the original sample before enrichment, the DNA of the sample after enrichment and the DNA of the deeply purified sample are respectively extracted, the DNA is diluted to the same concentration, PCR amplification and qPCR analysis are performed by using ITS as a primer, and the enrichment effect is verified by comparing the intensity and the specificity of PCR bands and the Cq value change of qPCR.
8. 8. Use of a method of enriching fungi from ruminant gastrointestinal contents according to any of claims 1-7 for the preparation of a kit for analyzing a fungal community from ruminant gastrointestinal contents.
9. 9. Use of the method of enriching fungi from ruminant gastrointestinal contents according to any of claims 1-7 for ruminant gastrointestinal fungal diversity analysis, quantitative detection or functional genomics research.

Description

Method for enriching fungi from ruminant gastrointestinal tract content and application thereof Technical Field The invention relates to the technical field of microbial separation, in particular to a method for enriching fungi from gastrointestinal tract contents of ruminants and application thereof. Background The gastrointestinal tract of ruminants (e.g., dairy cows) constitutes a complex and dynamic micro-ecological system that harbors a vast community of microorganisms including bacteria, archaea, protozoa, and fungi. The microorganisms and the host form a compact symbiotic relationship and are jointly involved in key physiological processes such as feed digestion, nutrient synthesis and absorption, energy metabolism, immune regulation and the like. Although advances in molecular biology have improved the knowledge of bacterial and archaeal communities, the search for gastrointestinal fungi has long been lagged. Such fungi, particularly anaerobic fungi (such as phylum Neocallimastigomycota), are considered to be one of the most efficient microbial populations for degrading plant lignocellulose. They break down complex plant cell wall polysaccharides by producing a powerful, diverse enzyme system that provides energy and substrates for hosts and microbial communities. Therefore, the method has important scientific and application values for optimizing nutrition of ruminants, developing novel feed additives and performing biomass conversion by utilizing an enzyme system of the ruminants. However, the primary technical bottleneck in accurately studying gastrointestinal fungi is their effective isolation and enrichment. Fungi have much lower biomass than bacteria in the intestinal content, and their mycelia often tightly intertwine and invade the inside of plant particles to form physical bonds, making it difficult to release target cells with simple washing. The sample also has a great amount of strong background interference such as bacteria, host cells, feed residues, free DNA and the like, and the ratio of the bacterial DNA in the total DNA directly extracted is generally overwhelming, so that the accuracy and the sensitivity of the subsequent fungus specific molecular analysis (such as ITS region PCR, qPCR and high-throughput sequencing) are seriously interfered. In the prior art, researchers have tried to deal with this problem by using a variety of methods, wherein the direct culture method has harsh conditions and limited representativeness, the general DNA extraction method cannot increase the proportion of fungal DNA in total DNA, the simple physical separation method is difficult to effectively distinguish fungi from impurities, and the single enzyme treatment method cannot cope with the complexity of the gastrointestinal content matrix. Therefore, a pretreatment method capable of efficiently and specifically enriching fungal cells and removing background interference to the maximum extent is needed in the art, which lays a foundation for the subsequent acquisition of high-purity fungal genome DNA and reliable community and function analysis. Accordingly, the present invention provides a method for enriching fungi from the gastrointestinal contents of ruminants and application thereof to solve the problems of the prior art. Disclosure of Invention Aiming at the problems, the invention aims to provide a method for enriching fungi from ruminant gastrointestinal tract contents and application thereof, which selectively enriches target fungi and furthest removes bacteria, host cells and physical impurities through a combined strategy of physical screening, composite enzymolysis and density gradient centrifugation, thereby laying a foundation for obtaining high-purity fungi genome DNA at the downstream and solving the problems that the high-purity fungi DNA cannot be obtained due to low fungi biomass, close combination with plant materials and serious bacterial background interference in the prior art, and further the accuracy of subsequent fungi molecular biology analysis is seriously affected. In order to achieve the aim, the invention is realized by the following technical scheme that the method for enriching fungi from the gastrointestinal contents of ruminants comprises the following steps: collecting fresh contents of different parts of the gastrointestinal tract of ruminants, and screening out a target part with highest fungus abundance through fungus specificity qPCR analysis; the content of the screened target part is physically screened, and then the large-particle impurities are removed by vacuum filtration, and the sediment is collected by centrifugation; Resuspending the centrifugally collected precipitate in a complex enzyme digestion solution for overnight digestion, wherein the complex enzyme digestion solution comprises cellulase, chitinase, pectase, glucanase, lysozyme, muramidase and DNaseI (deoxyribonuclease I) and is used for dissociating the combination of fungi and plant materi