Absolute quantitative method for microflora based on multi-internal standard system

Abstract

The invention discloses an absolute quantitative method for microflora based on a multi-internal standard system, which belongs to the field of biotechnology and omics analysis. The present inventionprovides a method for efficiently quantifying microbes during amplicon sequencing, the method is characterized in that the target plasmid with a corresponding gradient is added according to the copy number of the total amount of microbes in a sample, the steps of DNA separation, quality control, and sequencing are carried out to obtain an OTU classification unit, and the absolute number of corresponding microbes in the sample is calculated based on a proportional relationship between the amount of target substance added and the detection amount. The method can realize the absolute quantification of the sample to-be-tested, greatly improves the accuracy of the measurement result, and has the advantages of low cost and the like, and plays an important role in promoting the progress of a sequencing technology.

Description

technical field [0001] The invention relates to a method for the absolute quantification of microbial communities based on a multi-internal standard system, which belongs to the field of biotechnology and omics analysis. Background technique [0002] Amplicon (amplicon) is a nucleotide sequence after DNA or RNA amplification, which is a highly targeted method for analyzing gene variation in specific genomic regions. For example, the ultra-deep sequencing of the amplified fragment of a certain gene obtained by PCR amplification can effectively identify the variation and analyze its characteristics. Amplicon sequencing mainly includes 16S rDNA sequencing, 18S rDNA sequencing, ITS sequencing and target region amplicon sequencing. To reflect the differences between species in the classification of bacteria, fungi, and archaea in environmental samples, it has an important guiding role in the study of microbial composition in environments such as oceans, soils, and intestinal fec...

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Problems solved by technology

[0003] However, at present, due to the limitations of amplicon sequencing technology, relative abundance is usually used to express the composition of microorganisms, and there is a lack of characterization methods for the absolute content of microorganisms in the target environment, so that different target environment samples or the same sample in When comparing across samples or time gradients under different time conditions, it is easy to get wrong conclusions and cannot specifically reflect the specific succession law of the same microorganism under the time gradient. This is the most important problem facing amplicon sequencing at present.

The main reason is that there are different loss rates of microbial DNA in the extraction process of different environmental samples, as well as the preference in the amplification process, which makes it impossible to absolutely quantify the biomass in the sample during the amplicon sequencing process.

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