CN113777292B - Method for detecting and evaluating tetracycline environmental risk by using paramecium biomarker and IBR (intermediate frequency region) - Google Patents
Method for detecting and evaluating tetracycline environmental risk by using paramecium biomarker and IBR (intermediate frequency region) Download PDFInfo
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Abstract
The invention belongs to the technical field of biological monitoring and evaluation of environmental pollution of antibiotics, and particularly relates to a method for evaluating tetracycline environmental risk by utilizing paramecium biomarkers and IBR detection.
Description
Technical Field
The invention belongs to the technical field of biological monitoring and evaluation of environmental pollution of antibiotics, and particularly relates to a method for evaluating tetracycline environmental risk by utilizing paramecium biomarkers and IBR detection.
Background
The prior art reported methods for the biological detection of tetracyclines using test species, concentration ranges, parameters of detection and whether or not to use IBR for the integrated analysis are as follows:
for example, CN201110271631.3 is an evaluation method for soil environmental safety of transgenic insect-resistant rice, which uses a transgenic insect-resistant rice straw soaking solution to culture paramecium, and determines whether the transgenic rice has an influence on growth, propagation and the like of the paramecium by counting the density of the cultured paramecium cells; detecting the damage of the paramecium DNA by using a comet electrophoresis technology, and determining the damage condition of the insect-resistant transgenic Bt gene rice to the paramecium DNA by counting and analyzing the comet cell numbers of an experimental group, a negative control group and a positive control group; thereby judging whether the transgenic insect-resistant rice is safe to the soil environment.
CN202110224110.6 discloses a novel method for detecting environmental toxic substances by a combined biological method, which comprises the following steps: and collecting samples of the environment sample to be detected, and judging the types and contents of toxic substances in the environment where the detection samples are positioned according to the results of the three tests by a seed germination test, a fine drilling screw killing test and an aquatic micro animal community killing test. Trigonella Foenum Linn and Carnis gallus Domesticus of two families of the plant kingdom, and animals of three different phylum of the animal kingdom, namely Trigonella Foenum Linn of the Liu Shengzuan head snail family and rotifer of the class Odontopathy, and Caterpillar of the class Celloclass and Tenebrio Albae of the class Leptodermia are combined. The method is complex in evaluation method and increases analysis difficulty.
The main drawbacks of the prior art include:
(1) Species for developing tetracycline biological detection in the past are not sensitive enough or have higher cost, long period and low standardization degree;
(2) The types of the biomarkers used in the past are few, the qualitative quantity is more, and the quantitative quantity is not enough;
(3) The detection concentration range and the actual concentration of the environment are not strong in pertinence, and the practicability is low;
(3) The IBR comprehensive analysis and evaluation are lacking.
The IBR index method is widely applied to marine environment quality evaluation. The method has low requirements on the types and the quantity of the screened biomarkers, can correlate biological response with pollutants to perform causal effect analysis, and is widely applied.
The IBRv2 index method avoids the deviation of calculation results caused by different marker sequences when the first generation IBR index method draws a star chart, and can reflect the induction and inhibition effects of various biomarkers under different pollution conditions.
However, for the environmental concentration and environmental risk assessment of specific substances, a great deal of research is required to establish, and how to rapidly and accurately assess the environmental concentration and environmental risk of tetracycline is a problem to be solved by the invention.
Disclosure of Invention
In view of the problems existing in the prior art, the invention provides a method for evaluating the tetracycline environmental risk by utilizing the paramecium biomarkers and the IBR detection, and the tetracycline environmental concentration and the environmental risk are rapidly evaluated by utilizing the integration analysis of multiple biomarkers and IBR of sensitive indicator organisms.
The invention is realized by the following technical scheme:
a method for evaluating Tetracycline (TC) environmental risk using a paramecium biomarker and IBR detection, comprising:
(1) Culturing paramecium, selecting single cell for culture, and taking logarithmic growth phase individual for tetracycline detection;
(2) Selecting solutions of tetracyclines with different concentrations as a sample;
(3) Taking insect liquid with a certain density, after the sample to be detected is processed, washing cells by PBS, lysing cells by cell lysate, centrifuging, collecting supernatant, and respectively detecting by using biomarkers;
(4) Integrated biomarker analysis (IBR), the calculation steps are as follows:
(1) standardization: all biomarker data were normalized to y=log (X/X 0 ). Wherein X is the average value of the measurement results of each biomarker on each station; x is X 0 Biomarker values for control sites;
(2) homogenizing: the homogenization value of each marker at each station is Z= (Y-M)/S, wherein Y is the standardized value of each biomarker; m is the total average value over all stations; s is the standard deviation of the standardized value of each biomarker on all stations;
(3) assignment: calculating the deviation index a=z-Z for each biomarker at each site 0 Wherein Z is the homogenization value of each marker at each station; z is Z 0 The homogeneity value of the marker on the control station is;
(4) IBRv2 was calculated: the sum of absolute values of deviation indices |a| of all the markers at each station is calculated first, and divided by the number n of the biomarkers. The calculation formula is as follows:
as a preferred embodiment of the present invention, the step (1) includes: the method can collect fresh water body rich in plankton, pick single cells, establish a monoclonal culture system in a constant temperature and humidity incubator at 20-25 ℃ by wheat grain culture solution, perform hunger-induced joint reproduction, perform mass pure culture, and take individuals in logarithmic growth phase for tetracycline detection.
As a preferred embodiment of the present invention, the step (2) includes: the concentration range of the tetracycline is 10 ng-400 ng/L, and in the range, 5 detection concentrations of 10ng,50ng,100ng,200ng and 400ng are set according to the actual detection requirement.
As a preferred embodiment of the present invention, the step (3) includes: after the sample to be tested was treated for 24 hours with an insect solution having a density of 1000ind/ml, the cells were washed with PBS, lysed with a cell lysate (20mMTris,150mM NaCl,1%TritonX-100,1mM EDTA), centrifuged at 8000rpm at 4℃for 10min, and the supernatant was collected.
As a preferred embodiment of the present invention, the step (3) includes: the kit is selected from SOD kit, CAT kit, MDA kit, GSH-PX kit, PGK kit, ATPase kit, CYP450 kit, RP kit, and can detect biomarker.
As a preferred embodiment of the present invention, the step (4) includes: when a is greater than 0, it indicates that the marker is induced, and vice versa.
As a preferred embodiment of the present invention, the step (4) includes: the higher the IBRv2 value, the higher the tetracycline contamination level, the greater the biological contamination pressure impact.
As a preferred embodiment of the present invention, the evaluation method includes:
a significant increase in sod+rp and a significant decrease in mda+cat indicates the presence of 10ng/L TC;
a significant increase in cat+cyp450 and a significant decrease in mda+gsh-px+rp+pgk indicates an increase in concentration to 50ng/L;
the co-elevation of SOD+CYP450 and the co-elevation of GSH-PX+ATPase+PGK indicate that the TC concentration reaches 100ng/L;
significant increases in CAT alone indicated concentrations up to 200ng/L;
the individual rise in RP and the decrease in other biomarkers indicate that TC concentration has reached 400ng/L.
As a preferred technical solution of the present invention, in particular,
a significant increase in sod+rp (ai=2.6, 1.24) and a significant decrease in mda+cat (ai= -1.87, -0.15) indicates the occurrence of 10ng/L TC;
a significant increase in cat+cyp450 (ai=1.39, 2.07) and a significant decrease in mda+gsh-px+rp+pgk (ai= -2.27, -1.57, -1.09, -1.54) indicates an increase in concentration to 50ng/L;
a common elevation of sod+cyp450 (ai=1.3, 1.59) and a common depression of GSH-px+atpase+pgk (ai= -1.45, -1.8, -1.62), indicating a TC concentration of 100ng/L;
a significant increase in CAT alone (ai=1.67) indicated a concentration of 200ng/L;
a single increase in RP (ai=0.8) and a decrease in other biomarkers indicates that TC concentration has reached 400ng/L.
The beneficial effects of the invention compared with the prior art include:
1) A new method for detecting and evaluating the environmental concentration of tetracycline by utilizing single-cell biology multiple biomarker integration analysis (IBR) is established;
2) The method comprises the standardized culture of the indicated organism and the detection and IBR calculation of 8 biomarkers;
3) Biomarker combinations evaluation parameters for 5 concentrations of tetracycline are presented.
Drawings
FIG. 1 response of paramecium caudatum biomarkers to tetracycline
FIG. 2 analysis of biomarker and tetracycline concentration correlation
FIG. 3 IBR analysis of 8 biomarkers from paramecium caudatum
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples, but embodiments of the present invention are not limited thereto.
Example 1
1. Materials and methods
1.1 indicating organisms
Uropeda (Parameci μm ca. Mu.dat. Mu.m), ciliate, olibanum, orchidaceae, paramethyst. Widely distributed in the still water body with a plurality of organic matters. Is sensitive to environmental pollutant stimulus, and is therefore often used for evaluation and research of environmental pollutants.
1.2 cultivation of paramecium
The method can collect fresh water body rich in plankton, pick single cells, establish a monoclonal culture system in a constant temperature and humidity incubator at 20-25 ℃ by wheat grain culture solution, perform hunger-induced joint reproduction, perform mass pure culture, and take individuals in logarithmic growth phase for tetracycline detection.
1.3 setting of the concentration range for tetracycline detection
According to various literature reports and environmental monitoring reports, the experimental concentration of the tetracycline in water and soil environments and in biological detectable conditions is comprehensively summarized and screened, the concentration range is determined to be 10 ng-400 ng/L, and in the range, 5 detection concentrations of 10ng,50ng,100ng,200ng and 400ng are set according to actual detection requirements.
1.4 multiple biomarker assays
Taking insect liquid with the density of 1000ind/ml, after 24 hours of treatment of a sample to be detected, washing cells by PBS, lysing cells by cell lysate (20mMTris,150mM NaCl,1%Triton X-100,1mM EDTA), centrifuging at 8000rpm for 10min at 4 ℃, collecting supernatant, and respectively detecting biomarkers by using a SOD kit, a CAT kit, an MDA kit, a GSH-PX kit, a PGK kit, an ATPase kit, a CYP450 kit and an RP kit.
1.5 Integrated biomarker analysis (IBR)
The calculation steps are as follows:
(1) standardization: all biomarker data were normalized to y=log (X/X 0 ). Wherein X is the average value of the measurement results of each biomarker on each station; x is X 0 Is the biomarker value for the control site.
(2) Homogenizing: the homogenization value of each marker at each station is z= (Y-M)/S. Wherein Y is the normalized value of each biomarker; m is the total average value over all stations; s is the standard deviation of normalized values for each biomarker over all sites.
(3) Assignment: calculating the deviation index a=z-Z for each biomarker at each site 0 . Wherein Z is the position of each markerA homogenization value for each station; z is Z 0 Is the homogeneity value of the marker at the control station. When a is greater than 0, it indicates that the marker is induced, and vice versa.
(4) IBRv2 was calculated: the sum of absolute values of deviation indices |a| of all the markers at each station is calculated first, and divided by the number n of the biomarkers. The calculation formula is as follows:
the higher the IBRv2 value, the higher the tetracycline contamination level, the greater the biological contamination pressure impact.
2. Results
2.1 response of Single biomarkers to Tetracycline
Tetracycline treatment for 24 hours caused significant changes in various biomarkers in urospora cells (fig. 1).
SOD enzyme activity (fig. 1-a) increased significantly at 10ng,50ng,100ng and 200ng treatments;
MDA content (FIG. 1-B) was significantly reduced at concentrations of 10ng,50ng,100ng,200ng and 400 ng;
CAT enzyme activity (FIG. 1-C), increased significantly at 50ng,100ng and 200ng treatments, decreased significantly at 400ng treatments;
GSH-PX enzyme activity (FIG. 1-D) was significantly higher than the control at 10ng and significantly lower at other concentrations.
The enzyme activity of PGK (fig. 1-E), significantly lower than control group at 50ng,100ng and 400ng treatments, with no significant difference between 10ng and 200 ng;
ATPase activity (FIG. 1-F), at 10ng and 50ng, the enzyme activity was significantly higher than that of the control group, and at 100ng and 400ng, significantly lower than that of the control group;
cell RP content (FIG. 1-G), significantly higher than control at 10ng and 400ng, and significantly lower than control at 50ng and 200ng treatments;
CYP450 activity (FIG. 1-H), at 10ng,50ng,100ng treatment, enzyme activity was significantly higher than that of the control group, and at 400ng treatment, enzyme activity was significantly lower than that of the control group.
The above results indicate that any single biomarker is not sufficiently stable and uniform in response to tetracycline exposure, and that a multiple biomarker integrated evaluation system needs to be established.
2.2 biomarker correlation under different concentrations of tetracycline stress
To construct a multiple biomarker panel, the analysis of biomarker correlations at different concentrations was performed (fig. 2), and the results showed that: there is a cross-positive correlation between 8 biomarkers at different concentrations of tetracycline stress, with the most relevant other markers being 4 of SOD, CYP450, PGK, RP. Indicating that oxidative stress and exogenous drug metabolism, glycolysis, and ribosomal protein synthesis may be the primary biological mechanisms of urospora cauda against tetracycline stress. But only RP is positively correlated with the concentration dependence analysis. Under 24hTC stress, the response of these 4 markers can be used as proof of the presence of TC contamination.
However, the response of these markers is only a qualitative indication of the presence of TC contamination, but not a quantitative indication of the extent of TC contamination
2.3 multiple biomarker integration analysis (IBR)
The response of 8 biomarkers of paramecium to 5 tetracycline concentrations for 24h stress was analyzed using IBR integration and the results are shown in figure 3.
A significant increase in sod+rp (ai=2.6, 1.24) and a significant decrease in mda+cat (ai= -1.87, -0.15) indicates the occurrence of 10ng/L TC;
a significant increase in cat+cyp450 (ai=1.39, 2.07) and a significant decrease in mda+gsh-px+rp+pgk (ai= -2.27, -1.57, -1.09, -1.54) indicates an increase in concentration to 50ng/L;
a common elevation of sod+cyp450 (ai=1.3, 1.59) and a common depression of GSH-px+atpase+pgk (ai= -1.45, -1.8, -1.62), indicating a TC concentration of 100ng/L;
a significant increase in CAT alone (ai=1.67) indicated a concentration of 200ng/L;
a single increase in RP (ai=0.8) and a decrease in other biomarkers indicates that TC concentration has reached 400ng/L.
Table 2: biomarker combination representative value
Comparative example
Other species were selected: chironomus larvae and human hepatocytes were evaluated by the methods described above, and the experimental results are shown in Table 3 below, which are inferior to the effects of using paramecium.
Table 3: detection of tetracycline markers from other species and concentration ranges thereof
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (7)
1. A method for evaluating Tetracycline (TC) environmental risk using a paramecium biomarker and IBR detection, comprising:
(1) Culturing paramecium, selecting single cell for culture, and taking logarithmic growth phase individual for tetracycline detection;
(2) Selecting solutions of tetracyclines with different concentrations as a sample;
(3) Taking a certain density of insect liquid, after a sample to be detected is processed, washing cells by PBS, lysing cells by using a cell lysate, centrifuging, collecting supernatant, and detecting by using biomarkers respectively, wherein the detection kits comprise an SOD kit, a CAT kit, an MDA kit, a GSH-PX kit, a PGK kit, an ATPase kit, a CYP450 kit and an RP kit;
(4) Integrated biomarker analysis (IBR), the calculation steps are as follows:
(1) standardization: all biomarker data were normalized to y=log (X/X 0 ) Wherein X is the average value of the measurement results of each biomarker at each station; x is X 0 Biomarker values for control sites;
(2) homogenizing: the homogenization value of each marker at each station is Z= (Y-M)/S, wherein Y is the standardized value of each biomarker; m is the total average value over all stations; s is the standard deviation of the standardized value of each biomarker on all stations;
(3) assignment: calculating the deviation index a=z-Z for each biomarker at each site 0 Wherein Z is the homogenization value of each marker at each station; z is Z 0 The homogeneity value of the marker on the control station is;
(4) IBRv2 was calculated: the sum of absolute values of deviation indexes |A| of all the markers of each station is calculated firstly, and then divided by the number n of the biomarkers, and the calculation formula is as follows:
the evaluation method comprises the following steps:
a significant increase in sod+rp and a significant decrease in mda+cat indicates the presence of 10ng/L TC;
a significant increase in cat+cyp450 and a significant decrease in mda+gsh-px+rp+pgk indicates an increase in concentration to 50ng/L;
the co-elevation of SOD+CYP450 and the co-elevation of GSH-PX+ATPase+PGK indicate that the TC concentration reaches 100ng/L;
significant increases in CAT alone indicated concentrations up to 200ng/L;
the individual rise in RP and the decrease in other biomarkers indicate that TC concentration has reached 400ng/L.
2. The method of claim 1, wherein step (1) comprises: the method can collect fresh water body rich in plankton, pick single cells, establish a monoclonal culture system in a constant temperature and humidity incubator at 20-25 ℃ by wheat grain culture solution, perform hunger-induced joint reproduction, perform mass pure culture, and take individuals in logarithmic growth phase for tetracycline detection.
3. The method of claim 1, wherein step (2) comprises: the tetracycline concentration ranges from 10ng to 400ng/L, within which 5 detection concentrations of 10ng,50ng,100ng,200ng and 400ng are set.
4. The method of claim 1, wherein step (3) comprises: taking insect liquid with the density of 1000ind/ml, after the sample to be detected is treated for 24 hours, PBS (phosphate buffer solution) is used for cleaning cells, cells are lysed by cell lysate with the composition of 20mMTris,150mM NaCl,1%Triton X-100 and 1mM EDTA, and the cells are centrifuged at 8000rpm for 10min at 4 ℃, and the supernatant is collected.
5. The method of claim 1, wherein step (4) comprises: when a is greater than 0, it indicates that the marker is induced, and vice versa.
6. The method of claim 1, wherein step (4) comprises: the higher the IBRv2 value, the higher the tetracycline contamination level, the greater the biological contamination pressure impact.
7. The method of claim 1, wherein a significant increase in sod+rp, ai = 2.6, 1.24, and a significant decrease in mda+cat, ai = -1.87, -0.15, respectively, indicates the occurrence of 10ng/L TC;
significant increases in cat+cyp450, ai = 1.39, 2.07, and mda+gsh-px+rp+pgk, respectively, significant decreases, ai = -2.27, -1.57, -1.09, -1.54, respectively, indicating increases in concentration to 50ng/L;
sod+cyp450 co-elevation, ai = 1.3, 1.59, and GSH-px+atpase+pgk co-elevation, ai = -1.45, -1.8, -1.62, respectively, indicating TC concentrations up to 100ng/L;
CAT alone was significantly elevated, ai=1.67, indicating a concentration of 200ng/L;
an individual increase in RP, ai=0.8, and a decrease in other biomarkers indicates that TC concentration has reached 400ng/L.
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| WO2006077981A1 (en) * | 2005-01-21 | 2006-07-27 | Miyazaki Prefecture | High-throughput functionality evaluation method, program and apparatus |
| JP2008022762A (en) * | 2006-07-20 | 2008-02-07 | Miyazakiken Sangyo Shien Zaidan | Method, program and apparatus for evaluating high throughput function |
| CN103146805A (en) * | 2013-03-07 | 2013-06-12 | 中国水产科学研究院黄海水产研究所 | Shellfish monitoring method for ocean oil spill pollution base on integration biomarker method |
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