阅读说明：本技术 一种利用秀丽隐杆线虫检测发育毒性的方法 (Method for detecting developmental toxicity by using caenorhabditis elegans ) 是由 于振洋 艾芳婷 尹大强 孟祥周 于 2019-11-04 设计创作，主要内容包括：本发明提供了一种利用秀丽隐杆线虫检测发育毒性的方法,属于生态风险评价领域,本发明以24孔细胞培养板作为主要暴露环境,配制有特定坡度的NGM培养基,在其一侧对秀丽隐杆线虫卵进行受试物质暴露,利用秀丽隐杆线虫从虫卵到成虫的发育阶段对外界环境变化的敏感性,在另一侧采用大肠杆菌食物诱导线虫产生运动行为,根据该发育与运动的特征进行发育毒性的检测,甚至不需要显微镜的辅助即可直接观察,采用肉眼直接观察食物侧线虫的有无及多少,即可快速、定性判断受试物质的发育毒性,从暴露至指标检测的用时在24h左右,缩短了指标检测时间,并集肉眼观测与显微观测于一体,检测结果具有较高的准确性和可靠性。(The invention provides a method for detecting developmental toxicity by using caenorhabditis elegans, belonging to the field of ecological risk evaluation, wherein a 24-hole cell culture plate is used as a main exposure environment, an NGM culture medium with a specific gradient is prepared, a caenorhabditis elegans egg is exposed to a tested substance on one side of the culture medium, the sensitivity of the caenorhabditis elegans from the egg to the change of the external environment in the development stage of an adult is utilized, escherichia coli food is adopted on the other side to induce the nematode to generate movement behavior, the developmental toxicity is detected according to the characteristics of the development and movement, even the developmental toxicity can be directly observed without the assistance of a microscope, the presence or the amount of the nematode on the food side is directly observed by naked eyes, the developmental toxicity of the tested substance can be rapidly and qualitatively judged, the time from exposure to index detection is about 24 hours, the index detection time is shortened, and the macroscopic observation and the microscopic observation are integrated into a, the detection result has higher accuracy and reliability.)

1. A method for detecting developmental toxicity by caenorhabditis elegans, comprising the steps of:

a. preparing a bacterial liquid: culturing uracil-deficient escherichia coli by using a culture medium, obtaining thalli, and mixing the thalli with a sterile potassium solution to obtain a bacterial liquid with a specific concentration;

b. preparing caenorhabditis elegans egg liquid: preparing caenorhabditis elegans eggs by a synchronization method, and adding a sterile potassium solution into the caenorhabditis elegans eggs for dilution to obtain an egg solution with a specific concentration;

c. preparing a culture medium: preparing a culture medium by using a 24-hole cell culture plate, placing the 24-hole cell culture plate at an included angle of 12-15 degrees with a horizontal plane so as to form an inclined state of the bottom surface of a hole in the 24-hole cell culture plate, adding a liquid NGM culture medium into each hole of the 24-hole cell culture plate, and obtaining a plurality of solid NGM culture media with gradients after the liquid NGM culture medium is condensed; placing the 24-hole cell culture plate at an included angle of 18-20 degrees with the horizontal plane, dropwise adding the bacterial liquid obtained in the step a at the highest position of each solid NGM culture medium, keeping the angle of the 24-hole cell culture plate unchanged, placing the 24-hole cell culture plate in an incubator for 12-24 hours, taking out the 24-hole cell culture plate, and inactivating uracil-deficient escherichia coli in the bacterial liquid;

d. grouping: horizontally placing the 24-hole cell culture plate in the step c, dropwise adding the caenorhabditis elegans egg liquid prepared in the step b at the lowest part of each solid NGM culture medium, taking all the solid NGM culture mediums in each row of the 24-hole cell culture plate as a group, selecting one group as a blank control group, and taking the other five groups as exposure groups;

e. preparing a test solution: diluting a tested substance by adopting a sterile potassium solution to obtain a tested substance solution, and preparing one part of the tested substance solution corresponding to each exposed group;

f. and (3) observation calculation: dripping a test substance solution with a concentration corresponding to that of caenorhabditis elegans ovum solution in each exposed group, dripping a sterile K-solution with the same concentration into a blank control group, exposing for 2 hours at 20 ℃, then placing a 24-hole cell culture plate on a biological clean bench, uncovering and blowing for 15-20min, evaporating excessive water, then placing the 24-hole cell culture plate on a 20 ℃ condition for culture, observing the number of caenorhabditis elegans moving to the position of the bacterial solution dripping position on the solid NGM culture medium in the step c by naked eyes or a microscope after 22h, and calculating the ratio of the number of the caenorhabditis elegans at the position to the total number of the caenorhabditis elegans to obtain the nematode passing rate;

g. and (3) numerical comparison: carrying out linear fitting by taking the data of the rate of passage of the caenorhabditis elegans as an ordinate and the data of the concentration of the test solution as an abscissa, and obtaining the numerical values of a and k in a fitting equation of the form of y ═ ax + k, wherein x is the concentration of the test solution, y is the rate of passage of the caenorhabditis elegans, k is a constant, a is a coefficient, and the rate of passage of the caenorhabditis elegans is calculated by using the fitting equation to be 50%: the value of the concentration of the test substance solution when y is 0.5 is recorded as the median effect concentration: EC50, based on this EC50 value, compared the developmental toxicity of different test substances, the lower the EC50 value, the stronger the developmental toxicity.

2. The method for detecting developmental toxicity of caenorhabditis elegans as claimed in claim 1, wherein in step a, 2mL sterile potassium solution is added to the bacterial liquid in sequence for dilution until the total amount of bacteria in the bacterial liquid is 10⁹To 10¹⁰one/mL.

3. The method according to claim 1, wherein in step b, 2mL of sterile potassium solution is added to the eggs of C.elegans in sequence for dilution until the solution of C.elegans eggs has a concentration of 10-12/μ L.

4. The method of claim 1, wherein the excess water in the solid NGM medium is first extracted from the deepest part of the well plate with a sterile absorbent paper and then placed on a biological clean bench.

Technical Field

The invention relates to the field of ecological risk evaluation, in particular to a method for detecting developmental toxicity by using caenorhabditis elegans.

Background

With the progress of human science and technology and the continuous improvement of quality of life, more and more chemicals are used for people's life, and along with the continuous migration and transformation of a large amount of chemicals entering the environment, the chemicals can finally flow into water, soil and atmospheric environment again through various ways, and can be accumulated gradually along with the food chain, finally the health hazard is generated to human beings, and the interference is generated to the stability of an ecosystem. Therefore, the toxicity detection of chemicals is an important means for evaluating health risks and environmental hazards.

Caenorhabditis elegans (Caenorhabditis elegans) has the advantages of precise and short life cycle, small volume, easy culture and the like, takes bacteria as food and non-parasitic, has no harm to people, animals and plants, and has high safety. Caenorhabditis elegans is very sensitive to changes of external environment, and the caenorhabditis elegans can change biological indexes such as growth, development, motor ability and the like under environmental stress.

The toxicity detection of chemicals at present mostly adopts acute lethal toxicity indexes, the method generally adopts experimental conditions of high concentration and acute exposure, the experimental conditions are not in accordance with the actual conditions of low concentration and chronic exposure of the actual environment, the detection accuracy is relatively low, so the environmental significance of the toxicity effects of growth and development and the like under the condition of low concentration is gradually highlighted, meanwhile, the traditional method needs to transfer the indexes when performing subsequent index detection, the time consumed from the beginning of exposure, to the transfer operation and the subsequent index detection is generally about 36 hours, and the developmental toxicity biological detection method has the defects of long detection period and the like, and is difficult to popularize efficiently.

Disclosure of Invention

In view of the above problems in the prior art, the present application provides a method for detecting developmental toxicity using caenorhabditis elegans. The invention has short detection period, is close to the exposure condition of the actual environment, integrates the macroscopic observation and the microscopic observation, and has higher accuracy and reliability of the detection result.

The technical scheme of the invention is as follows:

a method for detecting developmental toxicity using caenorhabditis elegans, the method comprising the steps of:

a. preparing a bacterial liquid: culturing uracil-deficient escherichia coli by using a culture medium, obtaining thalli, and mixing the thalli with a sterile potassium solution to obtain a bacterial liquid with a specific concentration;

b. preparing caenorhabditis elegans egg liquid: preparing caenorhabditis elegans eggs by a synchronization method, and adding a sterile potassium solution into the caenorhabditis elegans eggs for dilution to obtain an egg solution with a specific concentration;

c. preparing a culture medium: preparing a culture medium by using a 24-hole cell culture plate, placing the 24-hole cell culture plate at an included angle of 12-15 degrees with a horizontal plane so as to form an inclined state of the bottom surface of a hole in the 24-hole cell culture plate, adding a liquid NGM culture medium into each hole of the 24-hole cell culture plate, and obtaining a plurality of solid NGM culture media with gradients after the liquid NGM culture medium is condensed; placing the 24-hole cell culture plate at an included angle of 18-20 degrees with the horizontal plane, dropwise adding the bacterial liquid obtained in the step a at the highest position of each solid NGM culture medium, keeping the angle of the 24-hole cell culture plate unchanged, placing the 24-hole cell culture plate in an incubator for 12-24 hours, taking out the 24-hole cell culture plate, and inactivating uracil-deficient escherichia coli in the bacterial liquid;

d. grouping: horizontally placing the 24-hole cell culture plate in the step c, dropwise adding the caenorhabditis elegans egg liquid prepared in the step b at the lowest part of each solid NGM culture medium, taking all the solid NGM culture mediums in each row of the 24-hole cell culture plate as a group, selecting one group as a blank control group, and taking the other five groups as exposure groups;

e. preparing a test solution: diluting a tested substance by adopting a sterile potassium solution to obtain a tested substance solution, and preparing one part of the tested substance solution corresponding to each exposed group;

f. and (3) observation calculation: dripping a test substance solution with a concentration corresponding to that of caenorhabditis elegans ovum solution in each exposed group, dripping a sterile K-solution with the same concentration into a blank control group, exposing for 2 hours at 20 ℃, then placing a 24-hole cell culture plate on a biological clean bench, uncovering and blowing for 15-20min, evaporating excessive water, then placing the 24-hole cell culture plate on a 20 ℃ condition for culture, observing the number of caenorhabditis elegans moving to the position of the bacterial solution dripping position on the solid NGM culture medium in the step c by naked eyes or a microscope after 22h, and calculating the ratio of the number of the caenorhabditis elegans at the position to the total number of the caenorhabditis elegans to obtain the nematode passing rate;

g. and (3) numerical comparison: carrying out linear fitting by taking the data of the rate of passage of the caenorhabditis elegans as an ordinate and the data of the concentration of the test solution as an abscissa, and obtaining the numerical values of a and k in a fitting equation of the form of y ═ ax + k, wherein x is the concentration of the test solution, y is the rate of passage of the caenorhabditis elegans, k is a constant, a is a coefficient, and the rate of passage of the caenorhabditis elegans is calculated by using the fitting equation to be 50%: the value of the concentration of the test substance solution when y is 0.5 is recorded as the median effect concentration: EC50, based on this EC50 value, compared the developmental toxicity of different test substances, the lower the EC50 value, the stronger the developmental toxicity.

In the scheme, the method comprises the following steps: in the step a, 2mL of sterile potassium solution is gradually added into the bacterial liquid for dilution until the total amount of bacteria in the bacterial liquid is 10⁹To 10¹⁰one/mL.

In the scheme, the method comprises the following steps: in the step b, 2mL of sterile potassium solution is added into the caenorhabditis elegans eggs to dilute the solution until the concentration of caenorhabditis elegans contained in the caenorhabditis elegans egg solution is 10-12 pieces/mu L.

In the scheme, the method comprises the following steps: in the step f, the excess water on the solid NGM culture medium is firstly absorbed at the deepest part of the pore plate by using sterile absorbent paper, and then the solid NGM culture medium is placed on a biological clean bench.

The beneficial technical effects of the invention are as follows:

(1) the NGM culture medium with a specific gradient is prepared, the caenorhabditis elegans eggs are exposed in a tested substance on one side of the NGM culture medium, the sensitivity of the caenorhabditis elegans from the eggs to the imago to the change of the external environment is utilized, escherichia coli food is adopted on the other side to induce the movement of the caenorhabditis elegans, a 24-hole cell culture plate is used as a main exposure environment, the exposure and index detection can be directly observed without transferring, even without the assistance of a microscope, the developmental toxicity of the tested substance can be rapidly and qualitatively judged by directly observing the existence and quantity of the nematode on the food side with naked eyes, the detection time of the subsequent index is about 24 hours, the detection time of the subsequent index is shortened, and the developmental toxicity of the tested substance can be further quantitatively judged by the subsequent microscopic observation;

(2) the culture medium with a specific shape is prepared, the same culture medium is used for completing the subsequent exposure and developmental toxicity detection, the transfer step between the exposure link and the index detection link in the traditional method is omitted, and the convenience of developmental toxicity detection is greatly accelerated. And based on the size of the larvae which can be identified, the existence of the nematodes can be directly observed by naked eyes, so that the development toxicity of the tested substance can be rapidly and qualitatively judged. In addition, the invention can further judge the proportion of the nematodes moving to the side with the food culture medium based on the counting observation of a microscope, and can quantitatively judge the developmental toxicity of the tested substance.

(3) The caenorhabditis elegans can provide a more reasonable data base which is closer to the actual environment exposure situation for the evaluation and management of the development toxicity of the chemical tested substances through the coexistence of food and the low-concentration exposure which are close to the actual environment, and further provide more reliable data for evaluating the ecological risk.

Drawings

FIG. 1 is a schematic diagram showing the shape of a specific NGM medium of the present invention and the results of the developmental toxicity of the test substance on C.elegans;

FIG. 2 is a schematic diagram showing the detection result of calcium chloride (mol/L) on the developmental toxicity of C.elegans.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings and examples. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.