CN107764965A - Combined pollutant biological genetic toxicity is quick in a kind of drinking water, the method for high flux detection - Google Patents
Combined pollutant biological genetic toxicity is quick in a kind of drinking water, the method for high flux detection Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/186—Water using one or more living organisms, e.g. a fish
- G01N33/1866—Water using one or more living organisms, e.g. a fish using microorganisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/1826—Organic contamination in water
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Abstract
The present invention relates to combined pollutant biological genetic toxicity in a kind of drinking water is quick, the method for high flux detection, belong to environment detection and evaluation technology category, it is the salmonella typhimurium by using 20 DEG C of preservations, cultivated 3 hours in recovery solution, poisonous substance exposure 3 hours is carried out directly in microwell plate after determining absorbance.CPRG is selected after reacting 30 minutes, to determine absorbance as enzymatic substrate.This method can be stablized to 0.015mgL to standard poisonous substance positive response‑1, standard deviation 0.00178mgL‑1.This method selects 20 DEG C of conditions to preserve bacterial strain, simplifies bacterial strain preservation steps, reduces fungi preservation difficulty, improves strain activity.Merge bacterial strain recovery and preceding incubation step and optimization reagent in experiment, will be limited in the used time within 8 hours, and improve sensitivity and stability.The present invention is especially suitable for high flux detection of the layman to water sample bio-toxicity, there is wide promotion prospect.
Description
Technical field
The present invention relates to combined pollutant biological genetic toxicity in a kind of drinking water is quick, the method for high flux detection, category
In environment detection and evaluation technology category.
Background technology
Drinking water is the necessity of the mankind, and water quality directly affects health.Synthesize or identified in the world at present
Chemical substance have more than 1,300 ten thousand kinds, it is common or have as many as 80,000 kinds.Wherein there is the material of carcinogenicity, it is such as polycyclic
The DBPs such as a variety of chemical pollutants and nitrosamine, nitrous acid amides such as aromatic hydrocarbons (BaP, coal tar etc.), aromatic amine
Deng content is extremely low in drinking water and exists with mixture state.The detection of single index based on chemical apparatuses has office
It is sex-limited, it is impossible to directly genetic toxic effect of the reaction to biology.Therefore, in drinking water combined pollutant bio-toxicity effect survey
It is scheduled on most important in water quality health risk assessment.
At present, mutagenicity test has Salmonella reversion test and umu by the national detection method for being included in standard such as ISO and De
Test.Salmonella reversion test is more using bacterial strain, and test operation is numerous and diverse, and operation requires rigorous aseptic, while can be disturbed by histidine, occurs
Doubtful positive findings.It is high to concrete water quality demand when carrying out genetoxic detection to water sample, cause water quality management cost to add
Greatly.Also without legal and quantitative description in terms of testing result.
Under comparing, concrete water quality used in traditional umu method of testings is few, it is not necessary to stringent sterilization condition, can use extinction
Degree measure analysis, it is simple to operate, it is easily quantitative, there is very high application feasibility.At present, this method is widely used for detecting water
The genetoxic of the samples such as body, soil, Atmospheric particulates.
But traditional umu method of testings still suffer from deficiency.- 80 DEG C of cryopreservation conditions of bacterial strain of conventional method, very
Conglomerate is unable to reach this requirement, significantly limit the application and popularization of method；Conventional method needs to answer bacterial strain
Soviet Union and preceding incubation, it is required close to 20 hours, extend the testing time；To bacterial strain specified in ISO13829 in international standard
Breaking-wall cell process is big using reagent volume, and species is more, easily causes experimental error；ONPG in method specified in international standard
For solution as enzymatic reaction substrate, testing result shows that cell is low to the positive response of toxicant, reduces experimental data
Accuracy；In addition, during emergent water quality monitoring, conventional method takes time and effort, and the toxicity high pass that can not tackle a large amount of samples measures
Examination.Therefore, develop that combined pollutant biological genetic toxicity in a kind of drinking water is quick, the method for high flux detection is very necessary.
The content of the invention
The purpose of the present invention is exactly to overcome -80 DEG C of cryopreservations of bacterial strain to be difficult to be widely popularized and traditional SOS/
Umu method of testing test periods are long, experiment reagent demand is big, experimental error is big, the deficiencies of being difficult to tackle large batch of sample, build
Stand that combined pollutant biological genetic toxicity in a kind of drinking water is quick, high-throughout method of testing, study -20 DEG C of Cord blood bacterium
Strain feasibility, Optimal Experimental reagent and condition, shorten the toxic exposure time, are limited to complete overall process detection in 8 hours.The invention
It is a kind of high sensitivity, quick, bulk, standardization detection water sample method, being capable of high flux detection sample biological heredity poison
Property.
Technical proposal that the invention solves the above-mentioned problems is as follows：Combined pollutant biological genetic toxicity high flux in drinking water
The method of detection, comprises the following steps：
40 μ L are taken to be stored in the S.typhimuriumTA1535/pSK1002 bacterial strains of -20 DEG C of conditions in 4mL TGA culture mediums
In, 3h is cultivated in 37 DEG C, carries out bacterial strain activation.The bacterial strain after 98 μ L activation is drawn into 96 microwell plates, by a series of concentration ladder
The 4-NQO standard liquids of degree sequentially add 2 μ L, carry out poisonous substance exposure.Microwell plate must carry out culture 3h under 37 DEG C, 1000rpm.
Under wavelength 570nm, A595 is determined.After culture terminates, 4mgmL is added-1CPRG solution, carry out enzymatic reaction.React 30min
Afterwards, stopped reaction, A570 is determined.By calculating the active size (RGA) of beta galactosidase, i.e.,Calculate and drink respectively
Water intoxication material and standard poisonous substance 4-NQO induction ratio (IR) are i.e.According to the IR values and 4-NQO of environmental sample
IR be compared, draw the 4-NQO of environmental sample equivalent concentration (TEQ4-NQO)：
Calculated according to the body weight 70kg of adult and daily water of drinking for 2L, combined pollutant biological heredity in drinking water
Toxicity carcinogenic risk evaluation method is：
Note：P values are more than 10-6, no carcinogenic risk；P values are in 10-6~10-4Between, water sample need to be controlled；P values are low
In 10-4When, there is carcinogenic risk.
The beneficial effects of the invention are as follows：Using having imported carrying in Salmonella typhimurtum S.typhimurium TA1535
The promoter and tetracycline and chloramphenicol resistance gene of umuC-Lac Z fusions, it is steady in two months under the conditions of realizing -20 DEG C
Surely bacterial strain is preserved, is advantageous to every profession and trade and promotes the use of.Using the bacterial strain as the acceptor of poisonous substance, optimize existing domestication, rear training
The method for supporting bacterium, shorten strain culturing time and toxicity detection time.Test tube method is replaced by Microdilution plate method simultaneously, will be limited
Within 8 hours, experiment flow simplifies for experiment, and the testing time shortens, high sensitivity, and accuracy is good.Meanwhile optimize reagent and use
It is cost-effective with other test conditions of experiment, operating efficiency is improved, detection water sample can be carried out in batches, is particularly used to drink
The detection of water, especially source water, the water quality of emergency state, quick and precisely reflect the state of water quality, be a set of quick, accurate
Exactness is high, high-throughout biological genetic toxicity method of testing.
Brief description of the drawings
The technology path schematic diagram of the high-throughout detection method of combined pollutant biological genetic toxicity in the drinking water of accompanying drawing 1.
The inventive method and conventional method inductivity are with 4-NQO change in concentration contrast curves in the implementation example of accompanying drawing 2.
Embodiment
For the purpose, technical scheme and advantage of the invention are more clearly understood, below in conjunction with specific embodiment, and reference
Accompanying drawing, the present invention is described in further detail.
The high-throughout detection method of combined pollutant biological genetic toxicity, the principle of institute's foundation in the drinking water of the present invention
For：PSK1002 specific plasmids, the plasmid are imported in umu experimental strain Salmonella typhimurtum S.typhimurium TA1535
Carry the drug resistant genes such as umu operators, umuD genes and the promoter and tetracycline of umuC-LacZ fusions and chloramphenicol.
When the bacterial strain produces DNA damage under external environment, thalline RecA gene outcomes are activated, and turn into active protease,
This enzyme can cut off aporepressor lexA, start umuC operators, and therefore cause the transcription of umuC-LacZ fusions, translation, table
Reach the fusion protein of betagalactosidase activity, the work that enzyme is induced is characterized by detecting the decomposition situation of specific substrates
Property, you can judge that tested thalline causes the degree of DNA damage, you can the biological genetic toxicity that detection is exposed in certain environment
Degree and size.In particular, in specific stage of reaction reaction product under specific wavelength 570nm and 595nm absorbance
(OD) it is different, the active size (RGA) of beta galactosidase is characterized by A570/A595.Drink is calculated by formula (1) and (2)
With the equivalent concentration and carcinogenic risk size of the biological genetic toxicity of combined pollutant in water.
The specific steps of the detection method of biological genetic toxicity include in the drinking water of the present invention：
Preparation standard poisonous substance 4-NQO gradient series
5mg4-NQO solid powders are weighed in 10mL DMSO, are fully mixed, until being completely dissolved, are configured to 500mg
L-1Solution.Draw 25 μ L500mgL-14-NQO solution fully mixes in 175 μ LDMSO, is configured to 62.5mgL-1's
4-NQO solution.20 μ L62.5mgL are taken successively-14-NQO solution continuous twice of dilution, is configured in 20 μ LDMSO solution
0mg·L-1、0.98mg·L-1、1.95mg·L-1、3.90mg·L-1、7.81mg·L-1、15.63mg·L-1、31.25mg·L-1
4-NQO standard liquids.
Draw 4-NQO standard curves
Each μ L of concentration standard liquid 2 are taken respectively into 98 μ L bacterium solutions, determine certain wave under various criterion concentration of poison respectively
Long lower A570 and A595 values.By the active size (RGA) for the beta galactosidase for calculating different disposal, i.e.,Pass through meter
The toxicant inductivity (IR) of various concentrations is calculated, i.e.,Fit standard curvilinear equation：IR=kC4-NQO+ b, obtain song
Line is as shown in Figure 1.
Drink the pretreatment of water sample and the detection of biological genetic toxicity
Water sample 2L is drunk to be filtered (if water sample is clean, this step can omit) with glass fiber filter.HLB solid-phase extraction columns are used
6mL methanol, 6mL ultra-pure waters activate successively, liquid is left naturally, and keep extraction column to moisten.Water sample is with 10mL/min stream
The fast solid-phase extraction column by having activated.After water sample all after, extraction is drained with nitrogen drying or solid-phase extraction device
Post.Solid-phase extraction column after drying is eluted with 8mL acetone, it is passed through solid-phase extraction column naturally.Eluent is in faint nitrogen
Drying is flowed down, and 20 μ L are settled to DMSO.The μ L of water sample 10 are added in 10 μ L DMSO after taking concentration, after mixing, same to line-marking method
Dilution 6 times, DMSO is blank control.Poisonous substance exposure experiment is carried out, determines absorbance, and then calculate according to formula (1) and (2)
4-NQO equivalent concentration and carcinogenic risk size in drinking water.
The positive response of the inventive method can be stablized to 0.015mgL-1, standard deviation 0.00178mgL-1.。
The measure of method precision
Using above method condition, standard series sample is prepared in experiment again every time, selects the linear model of identical graticule
Enclose.The measurement result of standard curve, as shown in table 1：
The standard curve data result of table 1
It is as follows that method test limit data are calculated by formula：
The method test limit tables of data of table 2
| Parallel sample (n >=7) | As a result |
| Average value (mg/L) | 0.015 |
| Standard deviation (mg/L) | 0.00178 |
| Relative standard deviation | 12.74% |
| Detection limit (mg/L) | ≤5 |
Particular embodiments described above, technical scheme and beneficial effect are carried out further specifically
It is bright, the specific embodiment of the present invention is the foregoing is only, is not used to the limit value present invention, it is all in the spiritual and former of the present invention
Within then, any modification equivalent substitution for being made, improvement etc., are within protection scope of the present invention.
Claims (8)
1. combined pollutant biological genetic toxicity is quick in a kind of drinking water, the method for high flux detection, it is characterised in that passes through
After selection is recovered using TGA solution to bacterial strain, the exposure of toxicant is carried out directly in microwell plate, before merging in experiment
Culture and resuscitation process, shorten experimental period, simplify experiment flow, reduce experimental cost.
2. combined pollutant biological genetic toxicity is quick, high flux detection in a kind of drinking water according to claim 1
Method, it is characterised in that S.typhimuriumTA1535/pSK1002 bacterial strains preserving type be the 10 times of dilutions of -20 DEG C of bacterial strains after
After 37 DEG C of culture 15.5h, with DMSO with 9:1 ratio is well mixed, and dispenses 1.5mL plastic tubes, in -20 DEG C of preservations, above-mentioned bar
Preserved under part and be used equally within 60 days combined pollutant biological genetic toxicity in drinking water to determine, using the bacterial strain of -20 DEG C of preservations, drop
Low preservation difficulty, experiment spending is saved, experiment sensitivity and stability has been improved, is advantageous to being widely popularized for technology.
3. combined pollutant biological genetic toxicity is quick, high flux detection in a kind of drinking water according to claim 1
Method, it is characterised in that recovery bacterium solution uses TGA solution.
4. combined pollutant biological genetic toxicity is quick, high flux detection in a kind of drinking water according to claim 1
Method, concentration of the 100 μ L4-NQO standard poisonous substance solution in micropore described in it are respectively：0mg·L-1、0.02mg·L-1、
0.039mg·L-1、0.078mg·L-1、0.156mg·L-1、0.313mg·L-1、0.625mg·L-1、1.25mg·L-1。
5. combined pollutant biological genetic toxicity is quick, high flux detection in a kind of drinking water according to claim 1
Method, it is characterised in that take 2 μ L to carry out toxicant exposure in 98 μ L bacterium solutions, above-mentioned toxicant carries with bacterium solution optimum proportioning
High experiment sensitivity, reduces test limit.
6. combined pollutant biological genetic toxicity is quick, high flux detection in a kind of drinking water according to claim 1
Method, it is characterised in that the μ L of bacterium solution 10 react 10min, used in experiment in 50 μ L BugBuster solution after drawing exposure
The agent of BugBuster Protein Extractions substitutes a variety of chemical reagent, not only simplifies experiment flow and operating procedure, and improve experiment
Sensitivity and stability.
7. combined pollutant biological genetic toxicity is quick, high flux detection in a kind of drinking water according to claim 1
Method, it is characterised in that the substrate selection CPRG of enzymatic reaction is enzymatic reaction substrate, using CPRG solution, improves whole detection
The sensitivity of process and bacterium solution are advantageous to improve the accuracy and reliability of experimental data to the positive response of standard poisonous substance.
8. combined pollutant biological genetic toxicity is quick, high flux detection in a kind of drinking water according to claim 1
Method, it is characterised in that toxic exposure process is carried out in 96 orifice plates, applies also for 384 orifice plates.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101570785A (en) * | 2009-06-10 | 2009-11-04 | 南京大学 | Method for detecting potential inherent toxicity of organic pollutants in water body |
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