Method for detecting polymorphism of flora of prawn culture water body

A technology for aquaculture water and polymorphism, which is applied in biochemical equipment and methods, microbial determination/inspection, DNA preparation, etc.

Inactive Publication Date: 2010-06-09
SOUTH CHINA UNIV OF TECH
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  • Claims
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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 t...

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  • Method for detecting polymorphism of flora of prawn culture water body
  • Method for detecting polymorphism of flora of prawn culture water body
  • Method for detecting polymorphism of flora of prawn culture water body

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Experimental program
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Embodiment 1

[0042] The aquaculture industry is an important pillar industry in my country's coastal areas, and the prawn aquaculture industry plays an important role in it. The area of ​​prawn aquaculture in my country reaches 2.4 million mu. However, due to the frequent occurrence of diseases, the output of shrimp farming in my country has been lower than the bottom in recent years. More and more scholars believe that the root cause of shrimp diseases is the deterioration of the farming ecological environment, and the vicious circle between the polluted environment and the farming organisms has led to the disease. continuous outbreak. However, only a part of the traditional method of isolating strains can be cultured, and when analyzing microbial diversity, it cannot accurately reflect the composition and diversity of the mixed flora. In this experiment, the shrimp breeding farm in Dongsheng Town, Zhongshan City, Guangdong Province was taken as the research object. T-RFLP combined with FI...

Embodiment 2

[0076] Use the shrimp culture water environment of Example 1 to carry out the flora detection and analysis at different time points, sampling once every two weeks from April to May (April 1st, April 16th, May 1st, May 16th). Collect 4 x 2000ml of water samples within 30cm of the surface at multiple points, bring them back to the laboratory for vacuum filtration, and store the filtrate at -20°C. The steps and process conditions for detecting the polymorphism of the shrimp aquaculture water are as follows:

[0077] (1) Extraction of microbial total DNA in water body samples of shrimp breeding farms:

[0078] a. Transfer the filtrate to a 100ml centrifuge tube, centrifuge at 13000rpm for 10min, suck off the supernatant, resuspend the precipitate with sterile water, and transfer to a 1.5ml centrifuge tube;

[0079] b. Add 567 μl TE, pipette repeatedly to resuspend it, add 40 μl 10% SDS and 5 μl 20mg / ml proteinase K, mix well, and incubate at 37°C for 1 hour;

[0080] c. Add 100 μ...

Embodiment 3

[0112] With embodiment 1 shrimp breeding water environment, T-RFLP combined with FISH technology directly detects the vertical (depth 40cm and 150cm) distribution changes of shrimp pond water microbial flora, on June 5, 2009 respectively in the surface layer (40cm) and deep layer (150cm) Random multi-point sampling once 2×2000ml, take it back to the laboratory for vacuum filtration, and store the filtrate at -20°C. The steps and process conditions for detecting the polymorphism of the shrimp culture water body flora are as follows:

[0113] (1) Extraction of microbial total DNA in the water body sample of the shrimp breeding farm;

[0114] a. Transfer the filtrate to a 100ml centrifuge tube, centrifuge at 12000rpm for 8min, suck off the supernatant, resuspend the precipitate with sterile water, and transfer to a 1.5ml centrifuge tube;

[0115] b. Add 567 μl TE, pipette repeatedly to resuspend it, add 35 μl 10% SDS and 4 μl 20mg / ml proteinase K, mix well, and incubate at 37°C f...

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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...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64C12N15/10
Inventor 林炜铁高利海张星朱雅楠罗剑飞
Owner SOUTH CHINA UNIV OF TECH
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