Water quality genotoxicity detection method based on semiconductor opening switch (SOS) effect of recombinant Escherichia coli

A technology for recombinant Escherichia coli and Escherichia coli, which is applied in the direction of material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of high price, insensitivity of chlorinated organic substances, and difficult access to recombinant bacteria system, and achieves simple and easy cultivation and operation , Improve the sensitivity of fluorescence detection and reduce the effect of detecting fluorescence background value

Active Publication Date: 2011-09-07
济南市供排水监测中心
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This system has the following problems in practical application: 1) It is not sensitive to chlorinated organic substances, which is not conducive to the detection of disinfection by-products in water bodies, especially in drinking water; Nearly 100 million people in the world have been infected, some of them have died, and there are microbial risks in the operation of the constructed Salmonella typhimurium system; 3) The application steps of the system are relatively cumbersome, and the detection of β-galactosidase activity requires the addition of substrates, Such as β-ONPG (o-nitrophenyl β-D-galactoside), the price is relatively expensive
4) Involving intellectual property rights, it is still difficult to obtain the recombinant bacterial system, and it is difficult to widely promote it in the domestic industry

Method used

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  • Water quality genotoxicity detection method based on semiconductor opening switch (SOS) effect of recombinant Escherichia coli
  • Water quality genotoxicity detection method based on semiconductor opening switch (SOS) effect of recombinant Escherichia coli
  • Water quality genotoxicity detection method based on semiconductor opening switch (SOS) effect of recombinant Escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The application of the genetic toxicity biological detection method based on the SOS effect of recombinant Escherichia coli in the detection of concentrated water samples (4L water concentrated to 1mL) of a water plant, the steps are as follows:

[0043] (1) Recovery and activation of recombinant Escherichia coli storage

[0044] Inject 200 μL of recombinant E. coli stock into 5 mL of modified LB liquid medium (tryptone 10 g / L, yeast extract 2.5 g / L, NaCl 10 g / L). In addition, glucose was added at 0.3% (v / w), ampicillin was added to a final concentration of 50 μg / mL, and cultured at 30°C with shaking at 150 rpm for 16-18 hours.

[0045] (2) Pre-preparation of E. coli detection solution

[0046] Add the resuscitation activation solution into fresh modified LB liquid medium at a volume ratio of 1 / 100, and culture it at 35-37°C with shaking at 180rpm until the OD (520nm) of the bacterial solution is 0.4, about 2 hours.

[0047] (3) Contact with the test sample

[0048] ...

Embodiment 2

[0062] Use the same method to detect the concentrated sample of water plant inflow water in Example 1 (4L water is concentrated to 1mL), and draw the ratio of fluorescence intensity to Cr 6+ Concentration standard curve, using the chromium ion concentration to represent the water quality toxicity of the water sample, the difference is: when resuscitating and activating, the culture temperature is 33°C, and the rotation speed is 180rpm; when preparing the detection solution, the rotation speed is 250rpm, and the cultivation is carried out until the bacterial liquid OD (520nm) 0.3; when the water sample to be tested or the chromium ion solution is in contact with the detection solution, the amount added is 20% of the volume of the detection solution, and the rotation speed is 250rpm. After testing, the fluorescence intensity ratio of the water sample to be tested is 1.90, which is brought into the obtained standard curve, and the genotoxicity of the water sample to be tested is e...

Embodiment 3

[0064] Use the same method to detect the concentrated sample of water plant inflow water in Example 1 (4L water is concentrated to 1mL), and draw the ratio of fluorescence intensity to Cr 6+ Concentration standard curve, using the chromium ion concentration to represent the water quality toxicity of the water sample, the difference is: centrifuge the E. coli contact liquid at a speed of 10,000rpm for 2min, and then break it by ultrasonic method For the bacterial cells, the procedure of crushing for 4 seconds, 4 seconds apart, and 15 times of crushing was adopted, and then the crushed cells were centrifuged at a high speed of 14000 rpm for 8 minutes. After testing, the fluorescence intensity ratio of the water sample to be tested is 2.08, which is brought into the resulting standard curve, and the genotoxicity of the water sample to be tested is equivalent to 0.0391mg / L of Cr 6+ effect.

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Abstract

The invention discloses a water quality genotoxicity detection method based on a semiconductor opening switch (SOS) effect of recombinant Escherichia coli. The method comprises the following steps of: recovering, activating and culturing recombinant Escherichia coli storage to obtain Escherichia coli detection solution; contacting a water sample to be measured with the detection solution, and simultaneously setting a contrast; centrifuging, ultrasonically disintegrating and performing other post-treatment on the contacted detection solution, and detecting a fluorescence intensity ratio of the water sample to be measured; measuring a fluorescence intensity ratio of the Cr6+ solution with different concentrations, which is contacted with the detection solution by using the same method; drawing a standard curve between the fluorescence intensity ratio and the Cr6+ concentration, and substituting the fluorescence intensity ratio of the water sample to represent the water quality toxicity of the water sample through the Cr6+ concentration. The method has no pathogenic risk, is easy and convenient to culture and operate, high in detection sensitivity and low in cost; the final result is represented by Cr6+ equivalent concentration, and can be compared with results of other biological detection methods; and the method is suitable for rapidly detecting genotoxicity of an environmental sample.

Description

technical field [0001] The invention relates to the technical field of genotoxicity detection of environmental pollutants, in particular to a method for detecting genotoxicity of environmental pollutants by using recombinant Escherichia coli carrying a reporter gene. Background technique [0002] The genetic toxicity analysis methods of environmental pollutants are divided into long-term tests and short-term tests. Because long-term test methods are time-consuming, laborious, and expensive for long-term maintenance of experimental animals, they have been gradually replaced by fast and cheap short-term screening methods. Short-term testing uses cytogenetic indicators to screen chemical mutagens, and usually uses biological cells such as plants, aquatic organisms, mammals, and microorganisms to monitor environmental mutagens. Currently, bacterial reverse mutations, micronucleus technology, and sister chromatids have been developed. Exchange test, SOS reaction test, single-cell...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 李力贾瑞宝孙韶华宋艳
Owner 济南市供排水监测中心
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