CN101149366A - Method for quickly quantifying eelworm death rate - Google Patents
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Abstract
This invention relates to a kind of new method which can fix quantify the death rate of the eelworm fast, and its application in the aspect such as medication filter, pesticide filter, environment evaluate, water quality evaluate and so on. This invention improves the traditional evaluate method of the eelworm death rate, after the eelworm being color stained, because the dead eelworm can be color stained, the live one can not, when the eelworm alkali dispels, it releases the coloring matter, the coloring matter has absorbency in certain wavelength, uses the absorbency value to reflect the death rate of the eelworm, and reflects the drug action, toxicity of the medication and the pesticide, and the change of the environment. So it can lighten the load that count the death eelworm in the microscope, the result is correct and fast, it can do high flux filter. The operation of this invention is simple, it has low request to the instrument, the cost is low, it has good application value.
Description
Technical field
The present invention relates to a kind of new method of fast quantification nemic death rate, and this method belongs to the application of model organism in the application of aspects such as drug screening, pesticide screening test, environmental evaluation, water quality assessment.
Background technology
Caenorhabditis elegans (C.elegans) is a kind of nematode that is grown in the soil, is food with the bacterium, is easy in laboratory cultures.Become the long 1mm of polypide, and entire body is transparent, is easy to observe at microscopically.L
3～L
4Phase is responsive to external world.From the adult that a development of fertilized ova becomes can lay eggs, it only needs 3 days.Under state of nature, Caenorhabditis elegans overwhelming majority individuality is a hermaphroditic, and can produce about 300 embryonated eggs its all one's life, if put several nematodes on a double dish, just can obtain a large amount of offsprings afterwards in several days
[1]Above-mentioned these characteristics make Caenorhabditis elegans aspect a lot of application be arranged all
[2]
People such as the Ke Ling of Xiamen University
[3]With the indicator organism of Caenorhabditis elegans, studied Cu as heavy metal pollution
2+, Cd
2+, Pb
2+, Zn
2+Deng the toxicity of 4 heavy metal species ion pair Caenorhabditis elegans, find that concentration of heavy metal ion is high more, toxicity is strong more, and nemic death rate is high more.The toxic reaction of nematode is rapider, dead with regard to occurring in a short time in the high concentration group; And in low concentration group, toxic reaction is slower, death just occurs through the long period, and control group is other each groups relatively, and the time-to-live is the longest.People such as the Yan Wen of Beijing Normal University
[4]Caenorhabditis elegans is used for the monitoring of ambient water quality, there is relation closely between same discovery Caenorhabditis elegans survival rate and the water sample toxicity, water sample toxicity is big more, nemic death rate is high more, and therefore the survival rate of available Caenorhabditis elegans is estimated drinking water resource and water factory's fresh water (FW) water sample toxicity.People such as Wang Xiao
[5]Also Caenorhabditis elegans is used for tracking to waste water of paper mill acid-base class poisonous substance.Experiment shows, has both reduced experimental cost with the harm of Caenorhabditis elegans indication heavy metal, water sample toxicity size etc., has improved detection efficiency again, so utilize its indicator organism as environmental evaluation, water quality assessment to have considerable application prospect.
People such as Wan Shuqing
[6]Mortality ratio with Caenorhabditis elegans is estimated gossypol, Nemacur, and Isofenphos methyl, omethoate, Methomyl, the insecticidal effect of Multiple Pesticides such as copper sulphate has been obtained desirable effect.Experiment shows: gossypol is better than other several nematode killing agents to the rate of knockdown of Caenorhabditis elegans, and Nemacur takes second place, and other several agricultural chemicals all can be clearly seen that from experimental result the rate of knockdown of Caenorhabditis elegans.This result is consistent with using silkworm moth as the result of study of experimental subjects
[7]The existing relevant article of research that Caenorhabditis elegans is used for anthelmintic drug medicine efficacy screening in the body is abroad delivered
[8], for the in vitro study of anthelmintic provides feasible method, and present good selectivity, accuracy and repeatability, and with low cost.These studies show that, the size of nemic death rate reacted for the drug effect of examination thing and the size of toxicity, therefore, nematode judgement anyway just becomes the important experimental index of this class research.
Judge that nematode classic method anyway is figure's method.Be that dead nematode is spasticity, and figure's bending of living more, generally coil or wriggle.But this method is inapplicable to being inactive and nematode palsy, and can only touch with platinum filament and pull out nematode and differentiate it anyway to cause that nematode moves this moment, therefore time-consuming, the effort of this method, and workload is very big.In order to change this situation, people such as Wan Shuqing
[6]Thereby attempt to distinguish the activity of nematode mensuration measured object nematode killing agent anyway by colouring method, they have compared dimethyl diaminophenazine chloride, methyl blue, the sarranine premium, the Color of 4 kinds of dyestuffs such as eosin finds that these 4 kinds of dyestuffs can both make dead nematode painted, and living nematode is not painted, and sarranine premium effect is best.But this method still need be counted one by one at microscopically, and workload is still very big.
United States Patent (USP) (patent No. is 20060191023) discloses a kind of fluorescent dye method of using and has differentiated the colouring method of nematode anyway.This method has been used for the screening study of anthelmintic drug, because the nematode that lives in the dyeing back does not have fluorescence, and dead nematode has fluorescence, utilize the size of testing fluorescence intensity to determine the mortality ratio of nematode, provide assurance for high flux screening like this, but this method is still higher to equipment requirements, is difficult to be able to promotion and application in common lab.
Because nematode self has weight, very fast sinking in aqueous solution is so there is another restriction point in the top fluorescence high-throughput screening method of carrying: promptly be difficult to grasp for the quantity of trying nematode in each hole in the porous plate.United States Patent (USP) (patent No. 20030154501) can make nematode be uniformly distributed in the solution with certain density PEG8000, polyvinyl alcohol (PVA), polyvinylpyrrolidone, glycerite, to guarantee that nematode evenly adds porous plate, makes for the examination nematode population controlled.But above-mentioned drug price is higher and nematode is had certain toxicity.
In view of the defective that prior art exists, the invention provides a kind of new method of fast quantification nemic death rate simple, with low cost, can be conveniently used in aspects such as new medicament screen, pesticide screening test, environmental evaluation water quality assessment.
List of references:
1. huge vast stretch of wooded country etc., the cultivation of Caenorhabditis elegans and store method, Zhejiang agricultural journal, 2007,19 (1): 34～36.
2. Qin Feng pine, Yang Chonglin, eelworm great discovery: the significant contribution of caenorhabditis elegans in life science, life science, 2006,18 (5): 419～424.
3. the beautiful rhabditis axei studies on acute toxicity of Ke Ling 4 heavy metal species ion pairs Xiamen University journal 2004,43 (6): 133～135.
4. nematode survival rate index such as Yan Wen is to the initial analysis Beijing Normal University journal 2005,41 (6): 616～619. of water sample relative toxicity
5. Wang Xiao etc. utilizes the model animal nematode to follow the trail of the research of acid-base class poisonous substance in the waste water of paper mill, environmental science, 2007,28 (4): 876～880.
6. ten thousand trees are blue or green, Zhou Qingchun etc., the research that nematicide determination of activity nematode is dyeed anyway and differentiates, agricultural chemicals, 1993,32 (1): 18～19.
7. Wang Sheng treasured, He Manli etc., several medicaments be to the test of pesticide effectiveness of ginkgo Io moth, northwest agricultural journal, 2000,9 (3): 43～46.
8.Charlemagne Gnoula et al.，5(6)～carboxyfluorescein diacetate as anindicator of caenorhabditis elegans viability for the development of an in vitroanthelmintic drug assay，sciencedirect 2007，71(5)：1886～1892..
Summary of the invention
The present invention mainly provides a kind of new method of fast quantification nemic death rate simple, with low cost, can use aspects such as new medicament screen, pesticide screening test, environmental evaluation, water quality assessment easily.Particular content is as follows:
To be experiment material, dispose the sucrose nematode liquid of 10%～30% concentration, preferred 18% sucrose solution through synchronized Caenorhabditis elegans.Draw the equally distributed nematode liquid of equivalent, add porous plate, add test sample then, treat the test sample effect after, nematode is dyeed with biological stain, the biological stain here refers to dimethyl diaminophenazine chloride, methyl blue, sarranine premium, eosin.After the dyeing, washing or damping fluid are washed till the solution clarification, use the NaOH alkaline hydrolysis nematode of 2.5mol/l then, with equimolar hydrochloric acid neutralization, make solution show neutral again, measure each hole absorbance log at the maximum wavelength place of dyestuff respectively.Dimethyl diaminophenazine chloride has absorption maximum at the 530nm place, and methyl blue is at the 668nm place, and the sarranine premium is at the 365nm place, and the absorption maximum of eosin is at the 513nm place.Measurement result shows the big or small proportional of the mortality ratio of nematode and measured OD value, and linear relationship is good, and the size of OD value has reflected the mortality ratio of nematode.
Prepare certain test sample concentration gradient, dispose certain density nematode liquid with 18% sucrose solution then, shift nematode in porous plate, the centrifugal sucrose that goes, wash 2～3 times, try nematode as standby supplying, the test sample adding is contained in the porous plate of nematode, place centrifugal treating after 4～48 hours, remove the residue test sample, every then pipe adds the neutral red solution of 100 microlitres 0.5%, dyeed 5～10 minutes, washing is to the solution clear, with 2.5mol/L sodium hydroxide solution alkaline hydrolysis nematode 5～10 minutes, the hydrochloric acid solution neutralization of volumetric molar concentration such as use then, in 530nm place mensuration absorbance log, utilize typical curve and regression equation with ultraviolet-visible pectrophotometer, calculate median lethal dose, thereby estimate the test sample acute toxicity.
Advantages such as method provided by the present invention is a kind of high-throughput screening method that is used for acute toxicity testing, and it is easy and simple to handle, and equipment requirements is simple just can be used in common lab, and this method can be applied to environmental evaluation, aspects such as agricultural chemicals and medicine.
Compared with former method, there is following advantage in the present invention:
(1) simple to equipment requirements, easy and simple to handle, the result is objective and accurate.
(2) Shi Yan high flux saves effort and time.
(3) expense is cheap, has good economic benefits.
Embodiment:
Embodiment one, nematode synchronization
Concrete grammar is as follows: the nematode that will cultivate 7～8d in double dish is used K solution (50mmol NaCl, 30mmol KCl, 10mmol NaAc) is flushed in the little vial, packing 1mL nematode liquid in each little vial, add 0.5mL sodium hypochlorite solution alkaline (2～4% chlorinated lime respectively, 0.4M NaOH), behind the abundant mixing, the centrifugal 4min of 3500rpm; After abandoning supernatant, with K liquid flushing 3 times, discard washing fluid after, nematode is layered on media surface uniformly.Place 16 ℃ of cultivations; After cultivating 2.5d, nematode grows L3～L4 phase basically, has at this moment finished the synchronization of nematode basically.
The uniformity coefficient of embodiment two, nematode
The uniformity coefficient of table 1. nematode in sucrose solution
| Hole 1 | Hole 2 | Hole 3 | Hole 4 | Hole 5 | Hole 6 | |
| Sucrose concentration 10% | Article 15, | Article 10, | Article 6, | Article 9, | Article 3, | Article 5, |
| Sucrose concentration 15% | Article 10, | Article 9, | Article 15, | Article 8, | Article 7, | Article 6, |
| Sucrose concentration 18% | Article 8, | Article 9, | Article 8, | Article 9, | Article 9, | Article 9, |
| Sucrose concentration 20% | Article 15, | Article 8, | Article 10, | Article 9, | Article 9, | Article 7, |
| Sucrose concentration 30% | Article 12, | Article 3, | Article 2, | Article 5, | Article 4, | Article 3, |
The distribution situation of nematode in table 2.18% sucrose solution
| Hole 1 | Hole 2 | Hole 3 | Hole 4 | Hole 5 | Hole 6 | |
| The A group | Article 6, | Article 11, | Article 8, | Article 7, | Article 6, | Article 7, |
| The B group | Article 9, | Article 7, | Article 7, | Article 8, | Article 10, | Article 11, |
| The C group | Article 11, | Article 8, | Article 9, | Article 9, | Article 8, | Article 9, |
| The D group | Article 9, | Article 8, | Article 9, | Article 10, | Article 8, | Article 11, |
| The E group | Article 11, | Article 7, | Article 9, | Article 10, | Article 10, | Article 9, |
In 10%～30% sucrose solution, 5 concentration of getting wherein are 10%, 15%, 18%, 20%, 30% distributes wherein nematode, sampling 100 microlitres are in 96 orifice plates after 5 minutes, every hole nematode number that counts, result such as table 1, last preferred 18% as making nematode be uniformly distributed in the preferred concentration (table 2) of solution.Statistical result showed, in 18% sucrose solution, F value=1.295 between each group, H according to d=0.05, is accepted in P value=0.299
0Think that the mean difference between each group does not have statistical significance, so think that the nematode that we prepared is uniform.
The foundation of embodiment three, typical curve
The concentration of nematode is 400/500 microlitres, and as for the amount of ordering for a trial nematode, this solution separated into two parts, a part is put to death nematode (solution 1) with heat treating process, and another part is untreated nematode solution (solution 2).Add water 200 microlitres respectively, 3000 rev/mins centrifugal 5 minutes, the washing twice, continue centrifugal, neutral red solution dyeing with 0.5% 8 minutes, wash the centrifugal excess dyestuff of removing,, the hydrochloric acid solution neutralization of volumetric molar concentration such as use then with 2.5mol/L sodium hydroxide solution alkaline hydrolysis nematode 9 minutes, centrifugal then 3 minutes, get supernatant, measure absorbance log at the 530nm place, obtain typical curve table 3.As shown below: canonical plotting, try to achieve equation of linear regression.
Table 3. typical curve
| Hole 1 | Hole 2 | Hole 3 | Hole 4 | Hole 5 | Hole 6 | |
| Solution 1 (μ l) | 100 | 300 | 500 | 700 | 900 | 1000 |
| Solution 2 (μ l) | 900 | 700 | Article 500, | 300 | 100 | 0 |
| After the dyeing | ||||||
| OD 530 | 0.225 | 0.482 | 0.576 | 0.721 | 0.892 | 0.999 |
Logarithm value with the number of dead nematode is a horizontal ordinate, is ordinate with the absorption value at 530nm place, and the regression equation of gained is Y=0.001X+0.180 R
2=0.9854
Embodiment four, the screening of agricultural insecticide effect
Coloring agent: it is standby that dimethyl diaminophenazine chloride, water are configured to 0.5% dyeing liquor.
For trying nematode: beautiful latent main line worm is configured to the nematode liquid of 800/500 microlitres with 18% sucrose solution
The configuration of medicine: thiram, get this product, use acetone solution, use tween～80 emulsifications then, add water and be configured to 15 mcg/ml, 10 mcg/ml, the solution for standby of 5 mcg/ml.
Line taking worm liquid 500 microlitres in the eppendorf pipe, add water centrifugal after, wash twice again, dosing 100 microlitres were placed 4 hours then, centrifugal after washing 2 times with dyeing liquor dyeing 8 minutes, is used alkali treatment then, with the acid neutralization, in 530nm place mensuration absorbance log.The results are shown in following table:
The screening of table 4. agricultural insecticide effect
| Drug concentration (ppm) | 20 | 15 | 10 | 5 | 0 |
| The OD value | 0.315 | 0.208 | 0.122 | 0.068 | 0.005 |
Regression equation is Y=0.0152X～0.0084 R
2=0.9804
Make Y=0.576, X=38.44ppm, promptly LC50 is 38.44ppm (1ppm=1 μ l/ml).
Embodiment five, environmental toxicity detect
Caenorhabditis elegans also can be survived under the nutrient solution condition, therefore also can be used as the research of aquatic toxicology, water pollution aspect, but rare to its research report as the Biological indicators kind of pollutant.Having very high exploitation with its foundation of carrying out high-throughout experimental technique is worth.The hydrobiont commonly used of the research of heavy metal pollution aspect is at present tested, with the acute toxicity test (table 5) of following 3 heavy metal species ion pair C.elegans, to understand the influence of heavy metal ion to its survival rate.By this test, the toxicologic study of the metal pair C.elegans that attaches most importance to provides with the practical basis of nematode as the high flux method of testing of heavy metal pollution.
Table 5. environmental toxicity detects
| Ionic type | Concentration (mg/l) | OD 530 |
| Copper ion (Cu 2+) | 18.1 | 0.417 |
| 9.02 | 0.218 | |
| Lead ion (Pb 2+) | 51.8 | 0.389 |
| 26.2 | 0.215 | |
| Zinc ion (Zn 2+) | 218 | 0.526 |
| 109 | 0.216 |
From the size of absorption value, draw the toxicity order of three metal ion species, Cu
2+＞Pb
2+＞Zn
2+, the result is consistent with bibliographical information.
The drug effect of embodiment six, pest repellant albendazole sheet detects
Albendazole sheet manufacturer: Sino-America Tianjin Shike Pharmaceutical Co., Ltd.; Specification: 0.2g/ sheet.
Coloring agent: it is standby that dimethyl diaminophenazine chloride, water are configured to 0.5% dyeing liquor.
For trying nematode: Caenorhabditis elegans is configured to the nematode liquid of 400/500 microlitres with 18% sucrose solution.
The configuration of medicine: get this product, with the glacial acetic acid dissolving, it is standby to be diluted to different concentration then, line taking worm liquid 500 microlitres are managed in eppendorf, add water centrifugal after, wash twice again, add test liquid 100 microlitres, placed 4 hours, centrifugal after washing 2 times with dyeing liquor dyeing 8 minutes, is used alkali treatment then, with the acid neutralization, measure absorbance log at the 530nm place.The results are shown in Table 6:
The acute toxicity of table 6. pest repellant albendazole sheet detects
| Drug concentration (ppm) | 400.0 | 200.0 | 100.0 | 50.0 | 0.00 |
| The OD value | 0.7524 | 0.326 | 0.247 | 0.058 | 0.012 |
Regression equation is Y=0.0018X+0.0024 R
2=0.9791 in order to calculating the median lethal dose of medicine, the theoretical foundation that the optimization and the screening of medicine provided.Make Y=0.576, get X=310.9ppm, promptly the MLC (median lethal concertration) of this medicine is 310.9ppm.
The toxicity test of embodiment seven tetracycline tablets
Tetracycline tablet manufacturer: Lanzhou pharmaceutical factory; Specification: 0.25g/ sheet.
Coloring agent: it is standby that dimethyl diaminophenazine chloride, water are configured to 0.5% dyeing liquor.
For trying nematode: Caenorhabditis elegans is configured to the nematode liquid of 400/500 microlitres with 18% sucrose solution.
The configuration of medicine: get this product, be dissolved in water, it is standby to be diluted to different concentration then, line taking worm liquid 500 microlitres are managed in eppendorf, add water centrifugal after, wash twice again, add certain density quadracycline solution 100 microlitres, placed 4 hours, centrifugal after washing 2 times with dyeing liquor dyeing 8 minutes, is used alkali treatment then, with the acid neutralization, measure absorbance log at the 530nm place.The results are shown in Table 7:
The toxicity test of table 7. tetracycline tablet
| Drug concentration (g/ml) | 0.250 | 0.125 | 0.0500 | 0.025 | 0.0125 |
| The OD value | 0.566 | 0.441 | 0.343 | 0.267 | 0.245 |
Getting regression equation is: Y=1.3276X+0.2496 R
2=0.9671.
Make Y=0.576, get X=0.246g/ml, promptly this MLC (median lethal concertration) is 0.246g/ml.
Claims (8)
1. the present invention is a kind of new method of fast quantification nemic death rate, it is characterized in that: 1. nematode can be the kind of free living, also can be the kind of parasitic life, wherein preferred Caenorhabditis elegans (Caenorhabditis elegans); 2. can obtain a large amount of nematodes by synchronization, perhaps obtain a large amount of nematodes by the hatching pieces of an egg; 3. nematode is uniformly distributed in porous plate, adds for test agent, dyes after the effect, and alkaline hydrolysis nematode released dye is measured the nematode alkali solution liquid OD value after neutralizing then; 4. the size of OD value has reflected the height of nemic death rate, thereby reflection is for the drug effect of test agent, toxicity etc.
2. the new method of a kind of fast quantification nemic death rate according to claim 1 is characterized in that this method can be applicable to the high flux screening of medicine effect.
3. the new method of a kind of fast quantification nemic death rate according to claim 1 is characterized in that this method can be applicable to the high flux screening of drug toxicity.
4. the new method of a kind of fast quantification nemic death rate according to claim 1 is characterized in that this method can be applicable to the high flux screening of pesticide efficacy.Estimate the action effect of the medicine of agricultural chemical insecticide
5. the new method of a kind of fast quantification nemic death rate according to claim 1 is characterized in that this method can be applicable to the high flux screening of toxicity of pesticide.
6. the new method of a kind of fast quantification nemic death rate according to claim 1 is characterized in that this method can be applicable to quick, the high flux evaluation of environmental pollution.
7. the new method of a kind of fast quantification nemic death rate according to claim 1 is characterized in that this method can be applicable to quick, the high flux evaluation of water quality.
8. the new method of a kind of fast quantification nemic death rate according to claim 1 is characterized in that this method can be applicable to fast, high flux judges that this is that the researcher in this field is to understand in the nematode scientific research anyway.
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| CN102213672A (en) * | 2010-04-03 | 2011-10-12 | 李红玉 | Method and kit for setting survival rate of nematode quickly |
| CN103913555A (en) * | 2014-04-15 | 2014-07-09 | 常州纺织服装职业技术学院 | Method for carrying out toxicity analysis on tail water of sewage treatment plant by using caenorhabditis elegans |
| CN105761244A (en) * | 2016-01-27 | 2016-07-13 | 中国科学技术大学 | Method, device and system for determining nematode mortality rate |
| CN106442488A (en) * | 2016-08-31 | 2017-02-22 | 威海中远造船科技有限公司 | Method for detecting living organisms with size of more than 50 micrometers in ballast water |
| CN113884012A (en) * | 2021-09-27 | 2022-01-04 | 海南大学 | Method for identifying survival state of plant pathogenic nematode larvae in activity determination of nematicide |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102213672A (en) * | 2010-04-03 | 2011-10-12 | 李红玉 | Method and kit for setting survival rate of nematode quickly |
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| CN103913555A (en) * | 2014-04-15 | 2014-07-09 | 常州纺织服装职业技术学院 | Method for carrying out toxicity analysis on tail water of sewage treatment plant by using caenorhabditis elegans |
| CN105761244A (en) * | 2016-01-27 | 2016-07-13 | 中国科学技术大学 | Method, device and system for determining nematode mortality rate |
| CN106442488A (en) * | 2016-08-31 | 2017-02-22 | 威海中远造船科技有限公司 | Method for detecting living organisms with size of more than 50 micrometers in ballast water |
| CN106442488B (en) * | 2016-08-31 | 2019-12-10 | 威海中远造船科技有限公司 | method for detecting living organisms with size of more than 50 mu m in ballast water |
| CN113884012A (en) * | 2021-09-27 | 2022-01-04 | 海南大学 | Method for identifying survival state of plant pathogenic nematode larvae in activity determination of nematicide |
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|---|---|
| CN101149366B (en) | 2015-03-04 |
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