Quantification method for denitrifying microorganisms in aquaculture environment sediments

A technology for denitrifying microorganisms and aquaculture, which is applied to the measurement/inspection of microorganisms, biochemical equipment and methods, etc. It can solve the problem of losing another type of microorganisms, and achieve short time, comprehensive and accurate quantitative information, and good results Effect

Inactive Publication Date: 2015-12-09
CHINA UNIV OF PETROLEUM (EAST CHINA)
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Problems solved by technology

However, the detection of a certain type of Nir gene alone will inevitably lose the information of another type of microorganisms, and cannot fully reflect the population composition and quantity of denitrifying microorganisms

Method used

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  • Quantification method for denitrifying microorganisms in aquaculture environment sediments
  • Quantification method for denitrifying microorganisms in aquaculture environment sediments
  • Quantification method for denitrifying microorganisms in aquaculture environment sediments

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Embodiment Construction

[0032] 1. Extraction of metagenomic DNA

[0033] Weigh 0.3 g of aquaculture water environment sediment sample, add it into a lysis reaction tube, and add 1100 μl of lysis buffer into the tube. Put the lysis reaction tube into the nucleic acid extractor, set the speed to 4.5, and the time to 30s for lysis. The centrifuge tube was centrifuged at 13000 rpm for 15 min at 4°C. The supernatant was transferred into a new 2ml centrifuge tube with a pipette tip cut off, and 250μl of protein precipitation buffer was added, mixed by gentle inversion 10 times, and centrifuged at 13000rpm at 4°C for 5min. Transfer the supernatant into two new centrifuge tubes with a tipped pipette, shake the suspension evenly to suspend it, and add 500 μl of the suspension to each centrifuge tube. Gently invert and mix for 2 minutes, and place the centrifuge tube in a rack at 4°C for 3 minutes. Aspirate 300 μl of supernatant from each tube and discard, and gently mix the supernatant and suspension. The...

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Abstract

The invention discloses a method for real-time fluorescence quantification PCR (qPCR) detection of denitrifying microorganisms in aquaculture environment sediments, and belongs to the technical field of environmental microbial ecology. The method comprises the following contents: metagenome DNA extraction and concentration determination of sediment samples in various stations, PCR amplification of nirK and nirS genes, construction of a standard plasmid, preparation of a standard curve, qPCR amplification and calculation of the number of denitrifying microorganisms in the samples according to a qPCR result in accordance with the standard curve. The method disclosed by the invention can be used for determining the number of the denitrifying microorganisms in an aquaculture environment through quantitative analysis on two genes, namely nirK and nirS, so as to overcome the shortcoming that 16S rRNA gene cannot be used in the quantification of the denitrifying microorganisms as well as the shortcoming that the prior art, which focuses on some functional gene only, is incomplete in quantification; and the provided method for detecting the number of the denitrifying microorganisms in the aquaculture environment is good in specificity, high in repeatability and high in accuracy. According to the method disclosed by the invention, logarithm values of nirK and nirS gene copy numbers keep a good linear relation with Ct value of a luminescence threshold, and the regression coefficient of the standard curve is above 0.99.

Description

Technical field: [0001] The invention relates to a method for real-time fluorescent quantitative PCR detection of denitrifying microorganisms in aquaculture environment sediments, belonging to the technical field of environmental microbial ecology, and the technology is used for measuring the quantity of denitrifying microorganisms in aquaculture sediments. technical background: [0002] my country is the world's largest aquaculture and aquatic product trading country. In 2012, the total amount of aquatic products reached 59.0768 million tons, accounting for about 38% of the global total, ranking first in the world for 24 consecutive years. In order to increase the unit output, excessive fertilizer and bait are often used in the breeding process, resulting in the continuous accumulation of residual bait, animal excrement and corpses in the breeding environment. disease or death. This not only reduces the output and quality of the aquaculture industry, but also pollutes the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2537/16C12Q2545/113C12Q2561/113C12Q2563/107
Inventor 李静刘建国杨杏娜肖梓军郗丽君赵静宜
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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