CN101149366B - Method for quickly quantifying eelworm death rate - Google Patents
Method for quickly quantifying eelworm death rate Download PDFInfo
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- CN101149366B CN101149366B CN200710202068.8A CN200710202068A CN101149366B CN 101149366 B CN101149366 B CN 101149366B CN 200710202068 A CN200710202068 A CN 200710202068A CN 101149366 B CN101149366 B CN 101149366B
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- Agricultural Chemicals And Associated Chemicals (AREA)
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 the application of this method in drug screening, pesticide screening test, environmental evaluation, water quality assessment etc., belong to the application of model organism.
Background technology
Caenorhabditis elegans (C.elegans) is the nematode of a kind of growth in soil, take bacterium as food, is easy in laboratory cultures.Become the long 1mm of polypide, and entire body is transparent, is easy under the microscope observe.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.In its natural state, Caenorhabditis elegans overwhelming majority individuality is hermaphroditic, and its all one's life can produce about 300 embryonated eggs, if put several nematodes on a double dish, just can obtain a large amount of offsprings after several days
[1].These features above-mentioned make Caenorhabditis elegans have application in a lot
[2].
The people such as Xiamen University Ke Ling
[3]using Caenorhabditis elegans as the indicator organism of heavy metal pollution, have studied Cu
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 higher, toxicity is stronger, and nemic death rate is higher.In high concentration group, the toxic reaction of nematode is comparatively rapid, just occurs death in a short time; And in low concentration group, toxic reaction is comparatively slow, just occur death through the long period, each relative to other group of control group, the time-to-live is the longest.The people such as the Yan Wen of Beijing Normal University
[4]caenorhabditis elegans is used for the monitoring of ambient water quality, relation is closely there is between same discovery Caenorhabditis elegans survival rate and Toxicity of Water Samples, Toxicity of Water Samples is larger, nemic death rate is higher, therefore can evaluate drinking water resource and water factory's fresh water (FW) Toxicity of Water Samples by the survival rate of Caenorhabditis elegans.The people such as Wang Xiao
[5]also Caenorhabditis elegans is used for the tracking to acid-base class poisonous substance in waste water of paper mill.Experiment display, indicates Heavy Metal Pollution, Toxicity of Water Samples size etc. both to reduce experimental cost with Caenorhabditis elegans, turn improves detection efficiency, so utilize its indicator organism as environmental evaluation, water quality assessment to have considerable application prospect.
The people such as Wan Shuqing
[6]evaluate gossypol by the mortality ratio of Caenorhabditis elegans, Nemacur, Isofenphos methyl, omethoate, Methomyl, the insecticidal effect of the Multiple Pesticides such as copper sulphate, achieve desirable effect.Experiment display: the rate of knockdown of gossypol to Caenorhabditis elegans is better than other several nematode killing agents, and Nemacur takes second place, and the rate of knockdown of other several agricultural chemicals to Caenorhabditis elegans all can be clearly seen that from experimental result.This result is consistent with using silkworm moth as the result of study of experimental subjects
[7].External research Caenorhabditis elegans being used for anthelmintic drug medicine efficacy screening in body has relevant article and delivers
[8], for the in vitro study of anthelmintic provides feasible method, and present good selectivity, accuracy and repeatability, and with low cost.These researchs show, the confession that the size of nemic death rate has been reacted tries the drug effect of thing and the size of toxicity, and therefore, nematode judgement anyway just becomes the important experimental index of this kind of research.
Judge that nematode classic method is anyway figure's method.Namely dead nematode is many in spasticity, and the stooped posture of living, generally coil or wriggle.But this method is to being inapplicable in nematode that is inactive and palsy, now can only touches with platinum filament and pulling out nematode it is anyway to cause nematode movement to differentiate, therefore this method time-consuming, require great effort, workload is very big.In order to change this situation, the people such as Wan Shuqing
[6]attempt distinguish life or death nematode by colouring method thus measure the activity of measured object nematode killing agent, they compare dimethyl diaminophenazine chloride, methyl blue, safranine, the Color of 4 kinds of dyestuffs such as eosin, finds that these 4 kinds of dyestuffs can make dead nematode painted, and living nematode is not painted, safranine effect is best.But this method still needs to count one by one under the microscope, workload is still very large.
United States Patent (USP) (patent No. is 20060191023) discloses a kind of colouring method applying fluorescent dye method to differentiate nematode anyway.This method is for the screening study of anthelmintic drug, due to the nematode unstressed configuration of living after dyeing, and the nematode of death has fluorescence, the size of test fluorescence intensity is utilized to determine the mortality ratio of nematode, like this for high flux screening provides guarantee, but this method is still higher to equipment requirement, be difficult to be able to promotion and application in common lab.
Because nematode self has weight, sink very soon in aqueous, so fluorescence high-throughput screening method carried above exists another restriction point: be namely difficult to grasp for the quantity of examination nematode in each hole in porous plate.United States Patent (USP) (patent No. 20030154501) certain density PEG8000, polyvinyl alcohol (PVA), polyvinylpyrrolidone, glycerite, nematode can be made to be uniformly distributed in solution, to ensure that nematode evenly adds porous plate, make for examination nematode population controlled.But above-mentioned drug price is higher and have certain toxicity to nematode.
In view of the defect that prior art exists, the invention provides a kind of new method of fast quantification nemic death rate simple, with low cost, the aspects such as new medicament screen, pesticide screening test, environmental evaluation water quality assessment can be conveniently used in.
List of references:
1. huge vast stretch of wooded country etc., the cultivation of Caenorhabditis elegans and store method, Zhejiang Agriculture 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. Ke Ling 4 heavy metal species ion pair Caenorthaditis elegans ws123 studies on acute toxicity Xiamen University journal 2004,43 (6): 133 ~ 135.
4. the 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., Land use models animal nematode follows the trail of the research of acid-base class poisonous substance in 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 life or death dyeing is differentiated, agricultural chemicals, 1993,32 (1): 18 ~ 19.
7. Wang Sheng is precious, He Manli etc., several medicament to the test of pesticide effectiveness of dictyoploca japonica butler, 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
Invention broadly provides a kind of new method of fast quantification nemic death rate simple, with low cost, the aspects such as new medicament screen, pesticide screening test, environmental evaluation, water quality assessment can be applied easily.Particular content is as follows:
With through synchronized Caenorhabditis elegans for experiment material, configuration 10% ~ 30% concentration sucrose nematode liquid, the preferably sucrose solution of 18%.The equally distributed nematode liquid of draws equal amounts, adds porous plate, then adds test sample, and after test sample effect, nematode biological stain, biological stain here refers to dimethyl diaminophenazine chloride, methyl blue, safranine, eosin.After dyeing, washing or damping fluid are washed till solution clarification, then use the NaOH alkaline hydrolysis nematode of 2.5mol/l, then neutralize with equimolar hydrochloric acid, make solution aobvious neutral, measure each hole absorbance log respectively at the maximum wavelength place of dyestuff.Dimethyl diaminophenazine chloride has absorption maximum at 530nm place, and methyl blue is at 668nm place, and safranine is at 365nm place, and the absorption maximum of eosin is at 513nm place.Measurement result shows that the size of the mortality ratio of nematode and measured OD value is proportional, and linear relationship is good, and the size of OD value reflects the mortality ratio of nematode.
Prepare certain test sample concentration gradient, then certain density nematode liquid is configured with the sucrose solution of 18%, transfer nematode is in porous plate, centrifugally remove sucrose, wash 2 ~ 3 times, examination nematode is supplied as subsequent use, test sample is added in the porous plate containing nematode, place centrifugal treating after 4 ~ 48 hours, remove residue test sample, then the neutral red solution adding 100 microlitres 0.5% is often managed, dye 5 ~ 10 minutes, washing, to solution clear, with 2.5mol/L sodium hydroxide solution alkaline hydrolysis nematode 5 ~ 10 minutes, then the hydrochloric acid solution neutralization of the volumetric molar concentration such as use, absorbance log is measured at 530nm place with ultraviolet-visible pectrophotometer, utilize typical curve and regression equation, calculate median lethal dose, thus evaluate test sample acute toxicity.
Method provided by the present invention is a kind of high-throughput screening method for acute toxicity testing, and the advantages such as it is easy and simple to handle, and equipment requirement is simple, just can apply in common lab, this method can be applied to environmental evaluation, the aspect such as agricultural chemicals and medicine.
Comparing with former method, there is following advantage in the present invention:
(1) simple to equipment requirement, easy and simple to handle, result is objective and accurate.
(2) high flux of testing, saves effort and time.
(3) low cost, has good economic benefit.
Embodiment:
Embodiment one, nematode synchronization
Concrete grammar is as follows: by nematode K solution (the 50mmol NaCl cultivating 7 ~ 8d in double dish, 30mmol KCl, 10mmol NaAc) be flushed in 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), fully after mixing, the centrifugal 4min of 3500rpm; After abandoning supernatant, rinse 3 times, after discarding washing fluid with K liquid, nematode is layered on media surface uniformly.Be placed in 16 DEG C of cultivations; After cultivating 2.5d, nematode grows L3 ~ L4 phase substantially, at this moment substantially completes the synchronization of nematode.
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 | |
| A group | Article 6, | Article 11, | Article 8, | Article 7, | Article 6, | Article 7, |
| B group | Article 9, | Article 7, | Article 7, | Article 8, | Article 10, | Article 11, |
| C group | Article 11, | Article 8, | Article 9, | Article 9, | Article 8, | Article 9, |
| D group | Article 9, | Article 8, | Article 9, | Article 10, | Article 8, | Article 11, |
| E group | Article 11, | Article 7, | Article 9, | Article 10, | Article 10, | Article 9, |
In the sucrose solution of 10% ~ 30%, get 5 concentration wherein that is 10%, 15%, 18%, 20%, 30% makes nematode distribute wherein, 100 microlitres are sampled in 96 orifice plates after 5 minutes, count every hole nematode number, result as table 1, finally preferably 18% as the preferred concentration (table 2) that nematode can be made to be uniformly distributed in solution.Statistical result showed, in 18% sucrose solution, F value=1.295 between each group, P value=0.299, according to α=0.05, accepts H0 and thinks that the mean difference between each group does not have statistical significance, so think that the nematode that we prepare is uniform.
The foundation of embodiment three, typical curve
The concentration of nematode is 400/500 microlitres, and as supplying the amount of ordering for a trial nematode, this solution is divided into two parts, and part heat treating process puts to death nematode (solution 1), and another part is untreated nematode solution (solution 2).Add water 200 microlitres respectively, centrifugal 5 minutes at 3000 revs/min, washing twice, continues centrifugal, neutral red solution with 0.5% dyes 8 minutes, wash and centrifugally remove excess dyestuff, with 2.5mol/L sodium hydroxide solution alkaline hydrolysis nematode 9 minutes, the then hydrochloric acid solution neutralization of the volumetric molar concentration such as use, then centrifugal 3 minutes, get supernatant, measure absorbance log at 530nm place, obtain typical curve table 3.As shown below: canonical plotting, try to achieve equation of linear regression.
Table 3. typical curve
With the logarithm value of the number of dead nematode for horizontal ordinate, with the absorption value at 530nm place for ordinate, the regression equation of gained is Y=0.001X+0.180 R
2=0.9854
Embodiment four, agricultural insecticide effect are screened
Coloring agent: dimethyl diaminophenazine chloride, be configured to water 0.5% dyeing liquor for subsequent use.
For examination nematode: the sucrose solution of beautiful hidden main line worm 18% is configured to the nematode liquid of 800/500 microlitres
The configuration of medicine: thiram, gets this product, with acetone solution, then uses tween ~ 80 emulsification, adds water and be configured to 15 mcg/ml, 10 mcg/ml, the solution for standby of 5 mcg/ml.
Line taking worm liquid 500 microlitre in eppendorf pipe, add water centrifugal after, then wash twice, dosing 100 microlitre, then place 4 hours, centrifugal after washing 2 times, dyes 8 minutes with dyeing liquor, then uses alkali treatment, with acid neutralization, in 530nm place mensuration absorbance log.The results are shown in following table:
Table 4. agricultural insecticide effect is screened
| Drug concentration (ppm) | 20 | 15 | 10 | 5 | 0 |
| 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, namely LC50 is 38.44ppm (1ppm=1 μ l/ml).
Embodiment five, environmental toxicity detect
Caenorhabditis elegans also can be survived under nutrient solution condition, therefore also can as the research of aquatic toxicology, water pollution aspect, but rarely reports its research as the Biological indicators kind of pollutant.With the foundation that it carries out high-throughout experimental technique, there is very high Development volue.The research of current heavy metal pollution aspect is commonly used hydrobiont to test, with the acute toxicity test (table 5) of following 3 heavy metal species ion pair C.elegans, to understand the impact of heavy metal ion on its survival rate.By this test, the toxicologic study of the metal pair C.elegans that attaches most importance to provides the practical basis of the high flux method of testing using nematode as 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+, result is consistent with bibliographical information.
The Composition analyzed of embodiment six, pest repellant albendazole tablet
Albendazole tablet manufacturer: Sino-America Tianjin Shike Pharmaceutical Co., Ltd.; Specification: 0.2g/ sheet.
Coloring agent: dimethyl diaminophenazine chloride, be configured to water 0.5% dyeing liquor for subsequent use.
For examination nematode: the sucrose solution of Caenorhabditis elegans 18% is configured to the nematode liquid of 400/500 microlitres.
The configuration of medicine: get this product, dissolves with glacial acetic acid, is then diluted to different concentration for subsequent use, line taking worm liquid 500 microlitre is managed in eppendorf, add water centrifugal after, then wash twice, add test liquid 100 microlitre, place 4 hours, centrifugal after washing 2 times, dyes 8 minutes with dyeing liquor, then uses alkali treatment, with acid neutralization, measure absorbance log at 530nm place.The results are shown in Table 6:
The acute toxicity of table 6. pest repellant albendazole tablet detects
| Drug concentration (ppm) | 400.0 | 200.0 | 100.0 | 50.0 | 0.00 |
| 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 calculate the median lethal dose of medicine, to the theoretical foundation that optimization and the screening of medicine provide.Make Y=0.576, obtain X=310.9ppm, namely the MLC (median lethal concertration) of this medicine is 310.9ppm.
The toxicity test of embodiment seven tetracycline tablet
Tetracycline tablet manufacturer: Lanzhou pharmaceutical factory; Specification: 0.25g/ sheet.
Coloring agent: dimethyl diaminophenazine chloride, be configured to water 0.5% dyeing liquor for subsequent use.
For examination nematode: the sucrose solution of Caenorhabditis elegans 18% is configured to the nematode liquid of 400/500 microlitres.
The configuration of medicine: get this product, is dissolved in water, and is then diluted to different concentration for subsequent use, line taking worm liquid 500 microlitre is managed in eppendorf, add water centrifugal after, then wash twice, add certain density quadracycline solution 100 microlitre, place 4 hours, centrifugal after washing 2 times, dyes 8 minutes with dyeing liquor, then uses alkali treatment, with acid neutralization, measure absorbance log at 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 |
| 0D value | 0.566 | 0.441 | 0.343 | 0.267 | 0.245 |
Obtaining regression equation is: Y=1.3276X+0.2496 R
2=0.9671.
Make Y=0.576, obtain X=0.246g/ml, namely this MLC (median lethal concertration) is 0.246g/ml.
Claims (9)
1. the new method of a fast quantification nemic death rate, described method is as follows, nematode is uniformly distributed in porous plate, add test sample, dye after effect, alkaline hydrolysis nematode released dye, then the nematode alkali solution liquid OD value after neutralization is measured, the measured size of OD value is directly proportional to the height of nemic death rate, thus reflects drug effect, the toxicity of test sample, described nematode be Caenorhabditis elegans (
caenorhabditis elegans), obtain described nematode by synchronization, or obtain described nematode by hatching pieces of an egg, wherein, described dyestuff is dimethyl diaminophenazine chloride, methyl blue, safranine, and eosin, is characterized in that: described nematode is the nematode liquid containing 10% ~ 30% sucrose.
2., according to the new method of a kind of fast quantification nemic death rate described in claim 1, it is characterized in that described nematode is the nematode liquid containing 18% sucrose.
3., according to the new method of a kind of fast quantification nemic death rate described in claim 1, it is characterized in that the method can be applicable to the high flux screening of medicine effect.
4., according to the new method of a kind of fast quantification nemic death rate described in claim 1, it is characterized in that the method can be applicable to the high flux screening of drug toxicity.
5. according to the new method of a kind of fast quantification nemic death rate described in claim 1, it is characterized in that the method can be applicable to the high flux screening of pesticide efficacy, evaluate the action effect of agricultural chemical insecticide.
6., according to the new method of a kind of fast quantification nemic death rate described in claim 1, it is characterized in that the method can be applicable to the high flux screening of toxicity of pesticide.
7., according to the new method of a kind of fast quantification nemic death rate described in claim 1, it is characterized in that the method can be applicable to quick, the high flux evaluation of environmental pollution.
8., according to the new method of a kind of fast quantification nemic death rate described in claim 1, it is characterized in that the method can be applicable to quick, the high flux evaluation of water quality.
9., according to the new method of a kind of fast quantification nemic death rate described in claim 1, it is characterized in that the method can be applicable to fast, high flux judges in nematode scientific research anyway.
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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 |
| 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 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1088046A (en) * | 1992-09-10 | 1994-06-22 | 联邦科学和工业研究组织 | The storage method of entomopathogenic nematodes |
| CN1305588A (en) * | 1998-06-19 | 2001-07-25 | 冷泉港实验室 | Detection of apoptotic cells in living nematodes |
| CN1645135A (en) * | 2005-01-21 | 2005-07-27 | 江西出入境检验检疫局检验检疫综合技术中心 | Rapid inspection for nematode larva of dormant pine wood |
| CN1951197A (en) * | 2006-11-01 | 2007-04-25 | 沈阳农业大学 | A novel method for controlling soybean root disease by using rhizobia |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1088046A (en) * | 1992-09-10 | 1994-06-22 | 联邦科学和工业研究组织 | The storage method of entomopathogenic nematodes |
| CN1305588A (en) * | 1998-06-19 | 2001-07-25 | 冷泉港实验室 | Detection of apoptotic cells in living nematodes |
| CN1645135A (en) * | 2005-01-21 | 2005-07-27 | 江西出入境检验检疫局检验检疫综合技术中心 | Rapid inspection for nematode larva of dormant pine wood |
| CN1951197A (en) * | 2006-11-01 | 2007-04-25 | 沈阳农业大学 | A novel method for controlling soybean root disease by using rhizobia |
Non-Patent Citations (2)
| Title |
|---|
| 曹博.应用RNAi技术研究cPTEN对线虫衰老的影响.《中国优秀硕士学位论文全文数据库(医药卫生科技辑)》.2007,(第4期),46-51. * |
| 鄢雯等.线虫存活率指标对水样相对毒性的初步分析.《北京师范大学学报(自然科学版)》.2005,第41卷(第6期),616-619. * |
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