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Method for quantitatively detecting biofilm gamma-proteobacteria

A quantitative detection method, a technology of proteobacteria, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of laborious, difficult to determine the diversity of microorganisms, and cannot satisfy the analysis of the relationship between the structure and function of microbial communities, etc. To achieve the effect of overcoming the slow speed

Inactive Publication Date: 2014-06-18
TONGJI UNIV
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  • Summary
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AI Technical Summary

Problems solved by technology

Furthermore, the culture-based microbial detection technology cannot meet the needs of analyzing the structure-function relationship of microbial communities due to its high selectivity, time-consuming, and labor-intensive shortcomings.
In addition, many organisms are not cultivable, and it is difficult to determine the microbial diversity in the ecosystem; traditional separation and counting techniques are also difficult to meet the requirements of dynamic monitoring of microbial community structure changes

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] (1) Recovery of γ-proteobacteria standard samples

[0043]Take out the standard sample of γ-proteobacteria stored at -80°C, dissolve it to room temperature, and mix thoroughly; take 20 μl of liquid standard sample of γ-proteobacteria into 2 ml of LB liquid medium (sterilize at 121°C for 25 minutes before using the medium) , placed in a constant temperature shaker at 37°C for 24 hours, and the rotation speed was controlled at 160rpm. After 24 hours, freshly activated standard bacterial liquids were obtained.

[0044] (2) Preparation of Escherichia coli Competent Cells

[0045] A. Select the Escherichia coli strain, pick a single colony from the LB plate and place it in 1ml of sterilized LB liquid medium, cultivate it on a constant temperature shaker at 37°C, and control the speed at 160rpm, and obtain fresh Escherichia coli after 24 hours Bacteria solution.

[0046] B. Fully mix the freshly cultured Escherichia coli liquid, take 400 μl of the liquid into 40 ml liquid ...

Embodiment 2

[0074] A quantitative detection method of biofilm gamma-proteobacteria, the method comprises the following steps:

[0075] The first step, pretreatment of standard γ-proteobacteria:

[0076] (1) Take out the standard sample of γ-proteobacteria stored at -80°C. After the standard sample of γ-proteobacteria reaches room temperature, add it to LB liquid medium, and culture it at 37°C for 24 hours to obtain recovered γ-proteobacteria. Sample;

[0077] (2) Preparation of Escherichia coli competent cells:

[0078] (a) Picking a single colony of Escherichia coli from the solid LB medium and placing it in a sterilized LB liquid medium, and cultivating it at a constant temperature at 37°C to obtain a fresh Escherichia coli liquid;

[0079] (b) Centrifuge the fresh Escherichia coli liquid at high speed at low temperature, remove the supernatant, then add 0°C sterilized calcium chloride, mix well, and let stand in loose ice for 30 minutes; Centrifuge at high speed for 5 minutes at ℃, ...

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Abstract

The invention relates to a method for quantitatively detecting biofilm gamma-proteobacteria. The method comprises the following steps: pretreating standard substance gamma-proteobacteria; pretreating a biofilm gamma-proteobacteria sample to be detected; carrying out real-time fluorescence quantitative PCR detection on the standard substance gamma-proteobacteria and making a standard curve equation y=-ax+b; then carrying out real-time fluorescence quantitative PCR detection on the biofilm gamma-proteobacteria to be detected to obtain a cycle threshold and obtaining the concentration of gamma-proteobacteria DNA in the sample according to the cycle threshold and a standard curve, wherein y is the cycle threshold and x is the logarithm of the concentration of DNA with 10 as the base number. Compared with the prior art, the method has the effects of effectively solving the problem that traditional pure culture methods have the defects of low analysis speed, more restrictive factors, low microbial diversity, low accuracy, strong subjective factors and the like, more scientifically monitoring the water treatment technology and the information of pathogenic bacteria in the pipe network biofilm, reflecting potential hazards in a drinking water system and providing more scientific bases for water quality purification of water supply networks.

Description

technical field [0001] The invention relates to a method for detecting bacteria, in particular to a method for quantitatively detecting biofilm γ-proteobacteria. Background technique [0002] In the field of water treatment and pipeline transportation, microbial indicators are an important part of water quality monitoring. The current microbial detection mainly focuses on suspended bacteria in water, but in fact, biofilms are formed in various water treatment processes and pipe networks, which has become a hidden danger to water quality stability. The formation of biofilm in the water supply system is mainly through the following five processes: (1) residual bacterial attachment; (2) the formation of microbial communities and the production of extracellular polymers; (3) the free expansion of the community to form a stable structure; (4) biological Membrane matures, new strains enter the biofilm to grow, organic and inorganic substances are combined to form a solution gradi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06
CPCC12Q1/686C12Q1/6851C12Q2561/113C12Q2545/114
Inventor 李伟英白涛沈程程李文明乔羽
Owner TONGJI UNIV
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