Methane/ ammoxidation bacterial community structure analysis method based on two-step method

Abstract

The invention belongs to the technical field of biological analysis, and in particular provides a method for simultaneous analysis of ammoxidation or methane oxidation correlated bacterial community structure. The method comprises the following steps: designing a high-degeneracy tag primer capable of covering a particulate methane oxidase alpha subunit gene and an ammoxidation monooxygenase alpha subunit gene; conducting PCR amplification on a genomic DNA extracted from an environmental sample by using the primer; conducting PCR amplification on the purified PCR products from the previous step by using a barcode marked tag primer; sequencing the amplified sequences; and analyzing to obtain ammoxidation or methane oxidation correlated bacterial community structure characteristics. The fidelity of the method is verified by different pmoA / amoA gene recombinant plasmid mixture experiments, so the method can significantly improve PCR amplification effect on the premise of improving primers coverage degree, and truly reflect the distribution of the different communities in environmental sample. The method completes structure analysis of ammonia oxidation and methane oxidation bacterial community in the environmental sample, and is applicable to structure analysis of other functional microflora.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a method that can be widely applied to the analysis of the structure of specific functional microbial communities in various ecological environment samples. Background technique [0002] Experimental methods relying on culture were once the main research methods of traditional microbiology. However, in environmental microbial samples, the number of microorganisms is usually large and the abundance of each kind is low. Most microorganisms are difficult to explore the culture conditions or even obtain pure culture. The mainstream method, and PCR for specific genes and analysis of sequence information is one of the important means. The conventional experimental method is to extract the total microbial genome DNA from environmental samples, and then perform PCR amplification for a certain gene, and obtain the sequence of the gene of various groups of microorganisms...

AI Technical Summary

Problems solved by technology

Due to the existence of degeneracy, the amplification efficiency of the primers is low, but a small amount of target gene fragments can still be amplified in the first few rounds of cycles, and both ends of the amplified fragments have lengthened the tag and the corresponding nucleotides of its complementary sequence

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