Method for detecting polymorphism of flora of prawn culture water body

Abstract

The invention discloses a method for detecting polymorphism of a flora of a prawn culture water body. The method comprises the following steps: (1) extracting total genomic DNA of mixed microbes in a prawn culture water body sample; (2) designing a specificity T-RFLP-PCR universal primer and performing PCR circulating reaction; (3) purifying a product and performing enzyme cutting on DNA by a specific restriction enzyme Hae III; (4) performing ionophortic separation on DNA segments by 1 percent agarose gel, performing fluorescent scanning on the DNA segments; (5) analyzing a polymorphism structure of a microbe flora; and (6) performing quantitative detection on predominant bacteria of the flora through fluorescence in situ hybridization technology. The method improves detection technology combining T-RFLP-PCR with FISH, uses fluorescence to mark a specific primer; and compared with the modern technology, the method has the characteristics of high repeatability, sensitivity, rapidness, accuracy, stability and the like, and can qualitatively and quantitatively analyze the ecological diversity of the microbes in the prawn culture water body along with time change and the dynamic change of a composite structure of the predominant flora.

Description

Technical field: [0001] The invention relates to a molecular detection method for the polymorphism of microbial flora in shrimp breeding water, belonging to the field of microbial ecology. Background technique: [0002] Microorganisms play an important role in many national economic production sectors, such as biomedical engineering (animal, human health care), biological pesticides (plant endophytes), light industry and environmental protection, aquaculture and other fields that use microorganisms for production activities. In most cases, the production activities of these sectors depend on the overall function of the microbial flora composed of a variety of microorganisms. For a long time, many industrial sectors that use microbial ecosystems for production activities have been unable to fully and objectively understand the community members in the system due to the limitations of traditional microbial isolation and culture techniques, let alone dynamically track the impac...

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Problems solved by technology

However, this molecular detection method can generally only analyze DNA fragments below 500 bp, and the information related to phylogenetic evolution is very little, and its map generally has only ten or twenty bands, and weak bacteria in the system cannot be detected. The requirements are relatively harsh, and the conditions of DGGE electrophoresis are different. Even the spectra obtained from the same sample are different. In the later stage, the gel is cut and recovered to establish a gene clone library, and then sequenced, so it is not fast, it consumes manpower and material resources, and when the microbial species is too high For a long time, the same band can contain multiple types of DNA information, therefore, it is impossible to analyze and compare the differences in the structure of microbial flora at a higher resolution

At present, RFLP technology is also based on 16S rRNA amplification. Total DNA is extracted from environmental samples, 16S rDNA is amplified with universal primers, and the amplified products are digested and analyzed with various restriction endonucleases. The composition of microorganisms in the group is different, and the enzyme digestion patterns are different, so that the DNA gel electrophoresis pattern reflecting the structure of the microbial flora can be obtained, but this method requires a variety of enzyme digestion processes, cloning, time-consuming, and only one Semi-quantitative or qualitative analysis, for groups with similar profiles, their composition is not necessarily the same

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