High-throughput absolute quantification method for three-domain microorganisms in soil

Abstract

The invention discloses a high-throughput absolute quantification method for three-domain microorganisms in soil. The method comprises the following steps that a three-domain universal internal standard strain E.coli WYL is re-suspended in sterile water to obtain a bacterial suspension with OD600 nm being 0.9-1.0; the three-domain universal internal standard strain is added into a soil sample; DNAin the soil sample is extracted; the three-domain universal internal standard strain in the sample is absolutely quantified; high-throughput sequencing of the soil sample is conducted; the absolute content of three-domain microorganisms is calculated. By means of the method, it is achieved that the absolute content of the three-domain microorganisms is represented, and therefore, the aim of cross-sample and cross-domain mutual comparison between the three-domain microorganisms can be achieved; the method provides a technical support for promoting community research on the three-domain microorganisms as a whole.

Description

technical field [0001] The invention belongs to the field of microorganisms, and relates to a method for realizing high-throughput absolute quantification of three-domain microorganisms (archaea, bacteria, fungi) based on high-throughput sequencing technology through the addition and utilization of three-domain general internal standard strains in samples method. Background technique [0002] Microorganisms are a general term for all tiny organisms that are difficult for individuals to observe with the naked eye. Because they have the characteristics of strong adaptability, easy variation, adaptability to various environments, and rich types, they have demonstrated huge development potential and are widely used in medicine, chemical industry, agriculture, Scholars from all walks of life, such as ecological environment, have paid attention. Studying microbial diversity and exploring the distribution pattern and change mechanism of microbial communities is of great significan...

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Problems solved by technology

[0008] 2. In a single sample, the high-throughput sequencing results for each domain can only characterize the dominance of different taxa in one domain in isolation, while different taxa belonging to the domains Archaea, Fungi and Bacteria (Taxon ) The domain collapse between the three domains is difficult to achieve

[0009] 3. When we try to compare the differences in the number of taxa in different domains in different samples, due to the differences in absolute totals between domains and samples, if the relative abundance is used for comparison and characterization, it may lead to erroneous results in conclusion

[0010] In order to overcome the shortcomings of relative abundance characterization in traditional high-throughput sequencing results, and to comprehensively study the relationship between microorganisms in the three domains and their responses to environmental changes based on microbial population ecology, it is urgent to develop a method that realizes the archaeal High-throughput absolute quantification technology for three-domain microorganisms, bacteria and fungi, realizes cross-sample cross-domain comparison of three-domain microorganisms with constant indicators (absolute content), makes up for the shortcomings of existing methods, and is useful for clarifying archaea, bacteria, and fungi in the environment. It is of great significance to elucidate the response mechanism of microorganisms to understand the biological changes in an all-round way and to further study microbial ecology.

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