阅读说明：本技术 一种地下水遗传毒性的检测方法及应用 (Detection method and application of groundwater genetic toxicity ) 是由 陈晓雯 赵建亮 胡立新 于 2019-10-10 设计创作，主要内容包括：本发明公开了一种地下水遗传毒性的检测方法及应用,属于环境健康遗传毒理学领域,包括步骤如下：预培养、前处理、暴露反应和结果分析,本发明通过在各个阶段进行适应性优化,提高了菌种敏感性；具有良好的平行性和重现性。本发明用于甄别和检测水环境具有的遗传毒性效应,评估遗传毒性效应强度,为进一步采取污染防治措施提供科学依据。(The invention discloses a detection method and application of groundwater genotoxicity, belonging to the field of environmental health genotoxicology, comprising the following steps: the method has the advantages that the method improves the sensitivity of strains by performing adaptive optimization at each stage; has good parallelism and reproducibility. The method is used for screening and detecting the genotoxic effect of the water environment, evaluating the strength of the genotoxic effect and providing scientific basis for further adopting pollution prevention and control measures.)

1. A method for detecting the genetic toxicity of underground water is characterized by comprising the following steps:

s1, pre-culture: adding Salmonella typhimurium TA1535/PSK1002 bacterial liquid into LB culture medium containing 50mg/mL ampicillin, and performing constant-temperature shaking overnight culture at 37 ℃ and 150rpm for 10-12h to obtain bacterial liquid for experiments;

s2, pretreatment: collecting an underground water sample, and treating the underground water sample by a solid phase extraction method to obtain an experimental sample;

s3, exposure reaction: adding the experimental bacterial liquid obtained in the step S1 into the experimental sample obtained in the step S2, diluting the experimental bacterial liquid by 10 times through TGA culture solution, carrying out constant-temperature shaking culture for 1.5-2h at 37 ℃ and 150rpm to enable the absorbance at 595nm to reach 0.5-0.8, carrying out constant-temperature shaking culture for 3.5-4h at 37 ℃ and 800rpm to obtain a diluted mixed liquid, and measuring the absorbance at 595 nm;

sequentially adding a B-buffer-SDS mixed solution and a 4.5mg/mL O-NPG solution into the diluted mixed solution, and carrying out oscillation reaction for 30-35min at 30 ℃ under the condition of 800 rpm; finally adding 1mol/L of Na₂CO₃Stopping the reaction by the solution, and measuring the absorbance at the wavelengths of 420nm and 550nm respectively;

s4, result analysis: and judging whether the sample has genetic toxicity or not by adopting the induction rate, further screening false positive results according to a gradient curve of the sample, and representing the genetic toxicity effect strength of the sample by using the toxicity equivalent concentration of the standard substance.

2. The method for detecting groundwater genotoxicity according to claim 1, wherein: the volume ratio of the bacterial liquid in the step S1 to the LB culture medium containing 50mg/mL ampicillin is 1: 400-420.

3. The method for detecting groundwater genotoxicity according to claim 2, wherein: the volume ratio of the bacterial liquid in the step S1 to the LB culture medium containing 50mg/mL ampicillin is 1: 420.

4. The method for detecting groundwater genotoxicity according to claim 1, wherein: and the absorbance of the experimental bacterial liquid obtained in the step S1 at 595nm is 1.6-1.8.

5. The groundwater genotoxicity detection method according to claim 1, wherein the solid phase extraction method in the step S2 specifically comprises: passing the collected underground water sample through a GF/F filter membrane, respectively activating an HLB solid-phase extraction column by using methanol and Milli-Q water, and then passing through the HLB column at the flow rate of 3-5 mL/min; after the completion, the sampling bottle is rinsed with 5 vt% methanol water solution, passes through an HLB column, and then Milli-Q water is added into each column respectively, and is dried for 2 hours; eluting with methanol and dichloromethane sequentially, mixing eluates, blow-drying under nitrogen, metering volume with methanol, filtering with 0.22 μm organic phase filter membrane, transferring into dark vial, and storing at-18 deg.C.

6. The method for detecting groundwater genotoxicity according to claim 1, wherein: the volume ratio of the experimental sample obtained in the step S2 in the step S3 to the experimental bacterial liquid obtained in the step S1 is 1: 18-20.

7. The method for detecting groundwater genotoxicity according to claim 1, wherein: the B-Buffer-SDS mixed solution in the step S3 was prepared by mixing the B-Buffer solution with 0.1 wt% SDS at a volume ratio of 20: 1.

8. The method for detecting groundwater genotoxicity according to claim 7, wherein: the volume ratio of the B-buffer-SDS mixed solution, the O-NPG solution and the diluted mixed solution in the step S3 is 11-14:2-4: 4-6.

9. The method for detecting groundwater genotoxicity according to claim 1, wherein: na in the step S3₂CO₃The volume ratio of the solution to the diluted mixed solution is 0.8-1.2: 0.8-1.5.

10. Use of the method of any one of claims 1-9 for the detection and assessment of genotoxicity.

Technical Field

The invention belongs to the field of environmental health genetic toxicology, and particularly relates to a detection method and application of groundwater genetic toxicity.

Background

In recent years, the overall quality of water environments has been degraded due to changes in chemical composition, physical properties and biological characteristics of groundwater caused by human activities, and studies have even pointed out that urban groundwater is seriously polluted in more than half cases. As an important source of drinking water for residents, the causes and the degrees of pollution of underground water are different, besides simply paying attention to the chemical concentration index of a certain compound, the comprehensive biological toxicity effect and the representation thereof are also worth paying attention to, and various toxicity detection technologies for underground water are urgently needed to be optimized and popularized, wherein detection on genetic toxicity effect is a typical example.

On the other hand, many studies have shown that there are many substances with genotoxic effects in environmental water and that prolonged exposure to these substances, even at low doses, can increase the incidence of cancer in humans. In addition, because of the wide variety of genotoxic substances, many concentrations are in trace or trace levels, and it is difficult to accurately evaluate the genetic effect by chemical detection means.

Genotoxic substances are primarily potential carcinogens which are electrophilic or form electrophilic intermediates after metabolic conversion. These substances can interact with DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) to cause damage variation of the DNA or RNA, damage is not repaired in time, mutation and even canceration can be caused, and harmful genetic variation effect can be generated in filial generation. The DNA damage comprises modes of DNA chain breakage, molecular matrix modification, DNA crosslinking and the like.

And part of polycyclic aromatic hydrocarbon, pesticide, phenols and anthraquinone substances can cause DNA damage of organisms, and have potential genetic effect. Some benzothiazole compounds also have significant toxic effects, particularly some heterocyclic amines.

However, at present, many researches on genotoxic substances in water environment, such as aromatic amine and nitro polycyclic aromatic hydrocarbon substances, are carried out abroad, and the researches on other compounds and the confirmation researches are relatively scattered. The domestic research on various genotoxic substances in water environment is less. Therefore, it is desirable to establish an appropriate screening method as soon as possible.

Disclosure of Invention

The invention aims to: the method is used for screening and detecting the genotoxicity effect of the water environment, evaluating the strength of the genotoxicity effect and providing scientific basis for further adopting pollution prevention and control measures.

The technical scheme adopted by the invention is as follows:

a method for detecting the genetic toxicity of underground water comprises the following steps:

s1, pre-culture: adding Salmonella typhimurium TA1535/PSK1002 bacterial liquid into LB culture medium containing 50mg/mL ampicillin, and performing constant-temperature shaking overnight culture at 37 ℃ and 150rpm for 10-12h to obtain bacterial liquid for experiments;

s2, pretreatment: collecting an underground water sample, and treating the underground water sample by a solid phase extraction method to obtain an experimental sample;

s3, exposure reaction: adding the experimental bacterial liquid obtained in the step S1 into the experimental sample obtained in the step S2, diluting the experimental bacterial liquid by 10 times through TGA culture solution, carrying out constant-temperature shaking culture for 1.5-2h at 37 ℃ and 150rpm to enable the absorbance at 595nm to reach 0.5-0.8, carrying out constant-temperature shaking culture for 3.5-4h at 37 ℃ and 800rpm to obtain a diluted mixed liquid, and measuring the absorbance at 595 nm;

sequentially adding a B-buffer-SDS mixed solution and a 4.5mg/mL O-NPG solution into the diluted mixed solution, and carrying out oscillation reaction for 30-35min at 30 ℃ under the condition of 800 rpm; finally adding 1mol/L of Na₂CO₃Stopping the reaction by the solution, and measuring the absorbance at the wavelengths of 420nm and 550nm respectively;

s4, result analysis: and judging whether the sample has genetic toxicity or not by adopting the induction rate, further screening false positive results according to a gradient curve of the sample, and representing the genetic toxicity effect strength of the sample by using the toxicity equivalent concentration of the standard substance.

LB culture solution: dissolving 21g LB broth in 1L milli-Q water, and sterilizing at 121 deg.C for 20 min; cooling in a clean bench, and adding 1mL of 50mg/mL ampicillin to obtain LB culture medium containing 50mg/mL ampicillin; stored at 4 ℃. Milli-Q Water ultrapure water was produced by Milli-Q Academic A10 ultrapure water system, Millipore Inc., USA. Is especially suitable for cell culture.

TGA culture solution: 10g tryptone, 5g NaCl and 11.9g HEPES buffer solution in 900mL milli-Q water, adjusting the pH value to 7.0 +/-0.2, diluting to 980mL milli-Q water, sterilizing at 121 ℃ for 20min, adding 20mL of 10 wt% glucose in a super clean bench, mixing uniformly, cooling, and adding 1mL of 50mg/mL ampicillin.

B-Buffer solution: 40.6g Na₂HPO₄·12H₂O(0.1M，MW＝358.14)，6.24gNaH₂PO₄·2H₂O(0.04M，MW＝156.01)，0.75gKCl(0.01M，MW＝74.55)，0.12g/0.25g MgSO₄/MgSO₄·7H₂O(0.001M，MW_MgSO4120.37) in 900mL milli-Q water. The pH value is adjusted to 7.0 +/-0.2, and the solution is diluted to 1000 mL. Before use, a solution of 40.5. mu.L of beta-mercaptoethanol was taken out in a fume hood (15 mL).

O-NPG solution: 45mg of O-NPG was dissolved in 10mL of phosphoric acid buffer solution, ready for use, and dissolved in a water bath (50 ℃). Wherein, the phosphate buffer solution: 0.61g NaH was weighed₂PO₄·2H₂O and 2.18g Na₂HPO₄·12H₂O, dissolving in 100mL milli-Q water, adjusting the pH value to 7.0 +/-0.2, and sterilizing for 20 min. O-NPG (2-Nitrophenyl beta-D-galactopyranoside, 2-Nitrophenyl-beta-D-galactoside, SIGMA) is used as color developing agent.

The principle of the invention is as follows: the Salmonella typhimurium TA1535/pSK 10020umuC gene is normally blocked by a repressor protein of LexA gene product, when environmental pollutants damage the DNA of bacteria, the bacteria generate SOS reaction, the RecA gene product of the bacteria is activated to become active proteolytic enzyme, the enzyme can cut off the repressor protein, the blocked umuC operon is started, and the umuC-LacZ fusion gene is driven to be transcribed and translated, so that the fusion protein with beta-galactosidase activity is expressed. By detecting the activity of the enzyme induced, the degree of DNA damage caused by the test substance can be determined.

Further, the volume ratio of the bacterial liquid in the step S1 to LB medium containing 50mg/mL ampicillin was 1: 400-420.

Further, the volume ratio of the bacterial liquid in the step S1 to LB medium containing 50mg/mL ampicillin was 1: 420.

Further, the absorbance of the experimental bacterial liquid obtained in the step S1 at 595nm was 1.6 to 1.8.

Further, the solid phase extraction method in the step S2 specifically includes: enabling the collected 1L of underground water sample to pass through a GF/F filter membrane, respectively activating an HLB solid-phase extraction column by using 10mL of methanol and 10mL of Milli-Q water, and then enabling the sample to pass through the HLB column at the flow speed of 3-5 mL/min; after the completion, the sampling bottle is rinsed with 2 × 50mL of 5 vt% methanol aqueous solution, passes through HLB columns, then 2 × 5mL of Milli-Q water is added into each column respectively, and is dried for 2 h; eluting with 7mL of methanol and 5mL of dichloromethane sequentially, mixing eluates, blow-drying under nitrogen, metering volume with 1mL of methanol, filtering with a 0.22 μm organic phase filter membrane, transferring into a dark vial, and storing at-18 ℃.

The GF/F filter membrane is a commercially available glass fiber filter membrane; Milli-Q Water ultrapure water was produced by Milli-Q Academic A10 ultrapure water system, Millipore Inc., USA. Is especially suitable for cell culture.

Further, the volume ratio of the experimental sample obtained in the step S2 in the step S3 to the experimental bacteria liquid obtained in the step S1 is 1: 18-20; preferably 1: 19. The mixing proportion of the sample and the bacterial liquid is crucial to the accuracy of the result; if the concentration of the bacterial liquid is too high, the sample is easy to dilute, the determination is difficult after the toxicity is reduced, and the gradient curve basically does not present inhibition characteristics; if the concentration of the bacterial liquid is too low, the growth amount of bacteria is insufficient, the bacteria are stimulated by a sample to die in a large amount, and the regularity is extremely poor; therefore, through experiments, the mixing ratio of the sample and the bacterial liquid is determined to be 1:19, the A595 value of the bacterial liquid is between 0.5 and 0.8, i.e. the total volume of the exposure stage is 200 μ L.

Further, the B-Buffer-SDS mixed solution in the step S3 was prepared by mixing the B-Buffer solution with 0.1 wt% SDS at a volume ratio of 20: 1; it is used as it is. SDS is sodium dodecyl sulfate.

Further, the volume ratio of the B-buffer-SDS mixed solution, the O-NPG solution and the diluted mixed solution in the step S3 is 11-14:2-4: 4-6; preferably 12:3: 5.

Further, Na in step S3₂CO₃The volume ratio of the solution to the diluted mixed solution is 0.8-1.2: 0.8-1.5; preferably 1: 1.

The method is applied to the detection and evaluation of genetic toxicity.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. the operation of the pre-culture stage of the invention ensures the activity of the strain, and adopts the LB culture medium, because the LB culture medium is beneficial to the rapid amplification of bacteria, and the strain activity is enhanced, the pre-culture requirement can be reached within 10-12 hours, and the problem that the prior art generally adopts the TGA culture medium to ensure that the strain can hardly reach the culture concentration requirement within the specified time is solved;

2. in the invention, in order to stabilize the growth environment of the strain in the experimental process, the TGA culture medium is used for carrying out constant-temperature oscillation culture on the experimental bacterial liquid for 1.5-2h under the conditions of 37 ℃ and 150rpm, the 595nm absorbance of the strain reaches 0.5-0.8 because the response value of the absorbance value response of the turbidity of the bacterial liquid at 595nm is the highest, in addition, the strain enters a logarithmic phase within 1.5-2h, so the pre-culture time is set to be 1.5-2h, and the strain sensitivity is high;

3. in the invention, the mixing ratio of the sample and the bacterial liquid is crucial to the accuracy of the result; if the concentration of the bacterial liquid is too high, the sample is easy to dilute, the determination is difficult after the toxicity is reduced, and the gradient curve basically does not present inhibition characteristics; if the concentration of the bacterial liquid is too low, the growth amount of bacteria is insufficient, the bacteria are stimulated by a sample to die in a large amount, and the regularity is extremely poor; therefore, through experiments, the mixing ratio of the sample and the bacterial liquid is determined to be 1:19, the accuracy is effectively improved;

4. in the invention, the exposure time is also a key control factor, the exposure time is short, incomplete exposure is easy to cause, the toxicity display is not obvious, the result regularity is not strong, a good S-type dosage relation cannot be presented, the exposure time is too long, and a false positive result is easy to appear, so that the exposure time is short, and the A of the bacterial liquid after the exposure stage is finished is used as a basis₅₉₅The value is changed, and the constant temperature oscillation condition of 37 ℃ and 800rpm is selected to be exposed for 4 hours, so that the accuracy is further improved;

5. the method not only adopts the conventional IR value to judge whether the sample has the genetic toxicity, but also further discriminates the false positive result according to the gradient curve of the sample, uses the equivalent concentration of the 4-NQO standard substance to represent the genetic toxicity effect strength of the sample, has good goodness of fit with the traditional method, and can reflect part of sample points with weak toxicity.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a diagram of groundwater genotoxic effect profile;

FIG. 2 is a graph of the growth curve (37 ℃, 175rpm) of Salmonella typhimurium TA1535/PSK 1002.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.

It is noted that relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

The features and properties of the present invention are described in further detail below with reference to examples.