CN101851659B - Ames experiment micro fluctuation method of mutagenicity detection for water bodies - Google Patents
Ames experiment micro fluctuation method of mutagenicity detection for water bodies Download PDFInfo
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Abstract
The invention relates to an Ames experiment micro fluctuation method of mutagenicity detection for water bodies, belonging to the technical field of microbes. The method of the invention is improved on the basis of the existing micro fluctuation detection method. In the method of the invention, under the optimal reaction conditions of TA97, TA98, TA100 and TA102 series of strains, a salmonella typhimurium TA98 strain is used as a test strain, and diamido fluorene is used as a positive matter; the experimental results of different strain solution concentration, different S9 concentration and different positive matter concentration in the system are analyzed through the experimental design, thereby determining the optimal reaction dose of the method; and the reaction conditions of TA97, TA100 and TA102 strains are analyzed, thereby verifying the universality of the reaction system. The method of the invention is used for mutagenicity detection of water quality, food and the like, has the advantages of high speed and simple and convenient operation, and has good application prospects.
Description
Technical field:
The present invention relates to a kind of Ames experiment micro fluctuation method of water body mutagenicity detection, belong to microbial technology field.
Background technology:
It is generally acknowledged that the chemical carcinogen of 85%-90% is a mutagen.Because the favorable reproducibility of Salmonella reversion test, prediction have found in rodent that the accuracy of carcinogenesis is higher.Thereby Salmonella reversion test is as the indispensable in the works part of any mutagenesis screening.
The fluctuation check of Green [1] in 1976 invention and traditional aberration inducing detect Salmonella reversion test and all return change as endpoint detection with bacterium, and in the test of known mutagenic compound, find that it can obtain the positive reaction result under lower concentration.Afterwards, Gatehouse was micromethod with the fluctuation check improvement, and this invention has not only alleviated workload greatly, and had further improved the susceptibility of fluctuation method.Domestic relevant for micro fluctuation method experimental system histidine concentrations, bacterial concentration, the report of biotin concentration [2].Aforesaid method does not have versatility between the Salmonella typhimurium different strains, and exists determinand to handle problems such as loaded down with trivial details.
Reference:
1.Green，MHL，WJ?M.Mutagen?testing?using?trp+reversion?in?Escherichia?coli.Mutat?Res?38(3)：32，1976.
2. long lamp recklessly, Zhan Zhuoling. influence factor and sensitivity study that micro fluctuation is tested. Hygiene Toxicology 4 (2): 115-116,1990.
Summary of the invention:
The objective of the invention is to overcome the deficiency of existing micro fluctuation detection method; And a kind of test system that is common to the Salmonella typhimurium different strains is provided, and make determinand handle the Ames experiment micro fluctuation method of the simple water body mutagenicity detection that becomes.
The inventive method is the improvement on existing micro fluctuation detection method basis.The inventive method has been set forth TA97, TA98, TA100, the optimum reaction condition of TA102 series bacterial strain.Be test strain through adopting Salmonella typhimurium TA98 bacterial strain specifically, the positive thing of diamino-fluorene is through experimental design; Different bacterial concentrations, different S9 concentration in the analysis system, the experimental result of different positive substrate concentrations has been confirmed the optimum response dosage of this method; And pass through TA97; TA100, TA102 bacterial strain response situation is analyzed, and has verified the versatility of reaction system.
The present invention has following several aspect with respect to existing micro fluctuation detection method improvement:
1, grown cultures liquid: vitamin H 0.8ug/ml, Histidine 0.2ug/ul, glucose 16mg/ml is dissolved in the Vogel-Bonner damping fluid.
2, purpurum bromocresolis (BCP): the 0.15g purpurum bromocresolis is dissolved in 30ml Vogel-Bonner damping fluid, makes the purpurum bromocresolis indicator of 5ug/ml, and the filtering with microporous membrane degerming is subsequent use.
3, bacterial concentration 10ul/ml in the reaction system, S9 concentration is 40ul/ml.
4, adopt determinand to expose bacterium 24 hours, add developer and continue to cultivate observations after 48 or 72 hours, promptly positive hole count.
5, with concentration be X-coordinate, the logarithm of positive hole count is the ordinate zou mapping, adopts conventional linear regression to divide fitting a straight line; The calculating of the linear equation through routine; With the slope is that slope is big more according to judgement determinand aberration inducing intensity, and promptly the mutagenesis ability is strong more relatively.
Method of the present invention is used for the mutagenicity detection of water quality and food etc., has quick, advantages of simple operation, and good prospects for application is arranged.
Description of drawings:
Fig. 1 is the positive thing reaction result of a various dose synoptic diagram.
Fig. 2 is reaction result synoptic diagram under the differential responses time.
Fig. 3 is a reaction result synoptic diagram under the different bacteria concentration conditions.
The practical implementation method:
1, bacterial strain:
With Salmonella typhimurium MOLTOX reference culture TA98, TA97, TA100, TA102, being cultured to bacteria concentration is 10
8
2, S9 cofactor mixed solution:
The preparation method accordings to: State Standard of the People's Republic of China-Salmonella typhimurium/Mammals microsomal enzyme test.
3,10%S-9 mixed solution (activation system):
Every 10ml contains aseptic 0.2mol/l phosphate buffered saline buffer (PH6.4) 6.0ml; 1.65mol/l Klorvess Liquid 0.2ml; 0.4mol/l magnesium chloride solution 0.2ml; 0.05mol/l G-6-P salts solution 1.0ml, 0.025mol/l coenzyme-II solution 1.6ml and rat liver homogenate liquid (S-9) 1.0ml, mixing.
4, solution:
Vogel-Bonner damping fluid stock solution (50 times): sodium hydrogen phosphate is pressed 16.5g, and Hydrocerol A 10.0g, potassium hydrogenphosphate 50.0g, sal epsom 1.0g, adding distil water are to 100ml, sterilizes in the dissolving back.
5, grown cultures liquid: vitamin H 0.8ug/ml, Histidine 0.2ug/ul, glucose 16mg/ml is dissolved in above-mentioned VB buffer.
6, purpurum bromocresolis (BCP):
0.15g purpurum bromocresolis is dissolved in 30ml Vogel-Bonner damping fluid, makes the purpurum bromocresolis indicator of 5ug/ml, the filtering with microporous membrane degerming is subsequent use.
7, positive thing: diamino-fluorene.
8, bacteria concentration 10ul/ml in the reaction system, S9 concentration is 40ul/ml.
9, above-mentioned reaction system is disposed the 12ml system with grown cultures liquid respectively, be added in 96 orifice plates, each concentration of each sample is 48 holes.Hatch 24h for 37 °.Every then hole adds purpurum bromocresolis (BCP) 20ul, continues to cultivate 72h, observes positive hole count.
10, culture is become yellow positive in the hole by purple; Being positive reaction, calculating the positive hole count of each concentration of every kind of bacterial classification, is X-coordinate with concentration; The logarithm of positive hole count is the ordinate zou mapping; Adopting conventional linear regression to divide fitting a straight line, through the calculating of conventional linear equation, is according to judging determinand aberration inducing intensity with the slope.Slope is big more, and promptly the mutagenesis ability is strong more relatively.
Fig. 1 shows: positive substrate concentration raises, and the positive findings hole count increases, and this phenomenon all has consistent result under different S9 concentration, draw our conclusion from three collinear slopes, and slope is high more, and the mutagenesis ability is strong more.
Fig. 2 shows: adopt determinand to expose after the bacterium 24 hours, add developer and continue to cultivate observations after 48 hours.According to time-course figure is set for different determinands the different reaction times.
Fig. 3 shows: different bacteria concentrations are to the influence of reaction result, and when bacteria concentration was got 10ul/ml, reaction result was relatively good, promptly are convenient to distinguish different positive thing mutagenesis intensity situation.
Through experiment showed, that above-mentioned experimental system is fit to TA97, TA98, TA100, the TA102 of Salmonella typhimurium.
Claims (1)
1. the Ames experiment micro fluctuation method of a water body mutagenicity detection is characterized in that:
A. grown cultures liquid: vitamin H 0.8ug/ml, Histidine 0.2ug/ul, glucose 16mg/ml is dissolved in the Vogel-Bonner damping fluid;
B. purpurum bromocresolis (BCP): the 0.15g purpurum bromocresolis is dissolved in 30ml Vogel-Bonner damping fluid, makes the purpurum bromocresolis indicator of 5ug/ml, and the filtering with microporous membrane degerming is subsequent use;
C. bacterial concentration 10ul/ml in the reaction system, S9 concentration is 40ul/ml;
D. adopt determinand to expose bacterium 24 hours, add the purpurum bromocresolis indicator and continue to cultivate observations after 48 or 72 hours, promptly positive hole count;
E. be X-coordinate with concentration, the logarithm of positive hole count is the ordinate zou mapping, adopts linear regression to divide fitting a straight line, through the calculating of linear equation, is that slope is big more according to judgement determinand aberration inducing intensity with the slope, and promptly the mutagenesis ability is strong more relatively.
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| CN102154486A (en) * | 2011-02-23 | 2011-08-17 | 南京大学 | Method for determining genetic toxicity of drinking water source water by applying RAPD-PCR (Random Amplified Polymorphic DNA-Polymerase Chain Reaction) |
| CN102321565B (en) * | 2011-09-06 | 2013-04-24 | 广东省微生物研究所 | Transgenic salmonella typhimurium TA1535/Pcda-GFP and construction method thereof |
| CN104962604B (en) * | 2015-07-31 | 2018-01-30 | 云南中烟工业有限责任公司 | Detection method for cigarette smoke condensates reverse mutation |
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Non-Patent Citations (4)
| Title |
|---|
| K Mortelmans,et al.《The Ames Salmonella/microsome mutagenicity assay》.《Mutation Research》.2000,第455卷(第1-2期),29-60. * |
| 中华人民共和国建设部.《城市供水水质检验方法标准及编制说明和研究报告》.《中华人民共和国城镇建设行业标准》.2001,全文. * |
| 夭建华.《国内外22个牌号卷烟烟气有害生物效应的比较研究》.《中国烟草学报》.2003,第9卷(第4期),12-19. * |
| 花日茂.《实验二 Ames实验(鼠伤寒沙门氏菌恢复突变试验)》.《环境毒理学》.2006,276-280. * |
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