Some standard content:
ICS 07. 100
National Standard of the People's Republic of China
GB15193.11-—2003
Replaces GB15193.11-1994
Sex-linked recessive lethal test for fruit flies
Sex-linked recessive lethal test2003-09-24Promulgated
Ministry of Health of the People's Republic of China
China Baojia Standardization Administration
Implemented on 2004-05-01bZxz.net
GB15193.11—2003
The full text of this standard is mandatory.
This standard replaces GB15193.11—1994 "Sex-linked recessive lethal test for fruit flies". Compared with GB15193.11-1994, this standard has the following major revisions: In the "Scope", the following are added: chemical, biological and physical factors that may cause harm to health during the production, processing, storage, transportation and sales of food; the test objects include food additives (including nutritional fortifiers), new food resources and their ingredients, new resource foods, irradiated foods, food containers and packaging materials, food tools, equipment, detergents, disinfectants, pesticide residues, veterinary drug residues, microorganisms used in the food industry, etc.;
"Dosage and grouping" is changed to a chapter parallel to "Operation procedures"; In the dose and grouping, "If the toxicity of the test substance is relatively low, the maximum dose of the test substance added to the feed can account for 5% of the feed. Positive controls can use ethyl methanesulfonate, methyl methanesulfonate, N-nitrosodimethylamine"; In the "Operation procedures", "Preparation of the test substance" is added. GB15193.11-1994 will be abolished from the date of implementation of this standard. This standard is proposed and managed by the Ministry of Health of the People's Republic of China. This standard was drafted by: Zhejiang Medical University, Shanghai Railway Medical College. The main drafters of this standard are: Xu Weian, Ye Enci, Zhu Huijuan, Li Shuguang. This standard was first issued in 1994 and this is the first revision. 78
1 Scope
Sex-linked recessive lethal test for fruit flies
This standard specifies the basic technical requirements for the sex-linked recessive lethal test for fruit flies. GB 15193.11—2003
This standard is applicable to the evaluation of the genetic toxicity of chemical, biological and physical factors that may cause harm to health during the production, processing, storage, transportation and sales of food. The test objects include food additives (including nutritional enhancers), new food resources and their ingredients, new resource foods, irradiated foods, food containers and packaging materials, food tools, equipment, detergents, disinfectants, pesticide residues, veterinary drug residues, microorganisms used in the food industry, etc.
2 Principle
2.1 Cross-inheritance of recessive genes in sex-linked inheritance, that is, the X chromosome of male flies is passed to female flies of the F generation, and then to male flies of the F2 generation through the F1 generation.
2.2 Recessive genes located on the X chromosome can be expressed in male flies under hemizygous conditions. Based on this, the eye color trait is determined by the genes on the X chromosome and is associated with the inheritance of the X chromosome as a marker for observing gene mutations on the X chromosome. Therefore, wild-type male flies (red round eyes, normal flies) are infected with poison and mated with Basc (Muller-5) female flies (light-colored stick eyes, with an inversion on each of the two X chromosomes to prevent the F1 generation from exchanging the treated paternal X chromosome with the maternal X chromosome). If the male flies are treated with the test substance, the gene on the X chromosome becomes recessive and lethal, then it can be expressed in the male flies of the F2 generation through the above two genetic rules, and the eye color trait is used as a marker to judge the results of the test. That is, according to Mendel's classification reaction, four F2 generations with different phenotypes are produced. When there is a recessive lethal trait, there are no male flies with red round eyes in the F2 generation. 3 Instruments and reagents
3.1 Instruments and equipment
Electrical constant temperature drying box, biochemical incubator, stereoscopic dissection microscope, magnifying glass, air conditioner, anesthesia bottle, milk bottle, large and small glass test tubes (3cmX8cm, 2cm×9cm), test tube tray and rack, white porcelain plate, sponge pad, brush, sponge plug. 3.2 Reagents
Ether, 75% ethanol, acetone, Tween.
3.3 Preparation of culture medium
Wash the large and small test tubes and sponge plugs, dry and disinfect them at 120℃ for 2h, and then set aside. 3.3.1 26g sucrose, 4g yeast powder, add 150ml water. 3.3.2 34g corn flour, 4g yeast powder, add 150ml water. 3.3.3 Steps: After boiling and dissolving according to 3.3.1, pour into 3.3.2 to mix and boil, then add 2mL of propionic acid, stir, and dispense into test tubes for later use.
4 Experimental animals
Drosophila. Male flies are wild-type Drosophila melanogaster (Drosophila melanogaster) aged 3 to 4 days, and female flies are virgin flies of the Basc (Muller-5) strain aged 3 to 5 days.
5 Dosage and grouping
Calculate the ICso or LDsn value according to the conventional method. Then, 1/2LCs or LD5o is the high dose, 1/5LDs, ~1/10LDs are the low doses, and set up negative (or solvent) and positive (2mmol/LMMS) control groups. If the test substance is less toxic, the maximum dose of the test substance added to the feed can account for 5% of the feed. Ethyl methanesulfonate, methyl methanesulfonate, and N-nitrosodimethylamine can be used as positive controls. 6 Operation steps
6.1 Preparation of test substances
Generally, the test substance should be dissolved in water. If the test substance is insoluble in water, it can be prepared into an emulsion or suspension with edible oil, medical starch, carboxymethyl cellulose, etc., and then diluted with water or saline before sampling. Avoid using dimethyl sulfoxide as a medium. The test substance should be freshly prepared before gavage, unless there is information indicating that it is stable when stored in a solution (or suspension, emulsion, etc.). 6.2 Collection of virgin flies
After the start of eclosion, remove all adult flies in the tube, and then the female flies collected within 6h to 12h are virgin flies. Put the virgin flies into a new test tube, with no more than 25 flies in one tube to avoid overcrowding. 6.3 Contact with test substance
Contact method of test substance is usually feeding with solution. After the test substance is dissolved, dilute it with 1% to 5% sucrose water to different concentrations, put a ball of toilet paper in the test tube, add 1ml of test solution to make the paper fully wet, put in male flies that have been starved for 4 hours for feeding, and when the newly prepared culture medium cools to 55℃, pour the test substance, stir rapidly with magnetic stirring for 2 minutes, and contact the test substance for 1 day to 3 days. 6.4 Mating procedure and method
In order to detect which stage of reproductive cells the test substance is most sensitive to, male flies are mated with virgin flies at intervals of 2-3-3 days after contact with the test substance (representing the effects on sperm, spermatocytes and spermatocytes respectively). That is, in each test tube, a treated male fly is mated with two virgin flies in sequence according to the above procedure, and then the F1 generation is mated with female and male (1:1 or 1:2) for F to F2. The F2 generation is observed after 12 to 14 days, and the incubation temperature is 25℃.
Each experimental group should have at least 3000 samples. 7 Data processing
The lethality is calculated based on the number of tested chromosomes (i.e., the number of female flies mated in the F, generation minus the number of infertile and waste tubes) and the number of lethal positive tubes. Number of lethal tubes
Lethality (%) = main
×1000
number of tested chromosomes
The lethality of the experimental group and the control group is statistically analyzed according to the KastenbaumandBowman method. 8 Result judgment
The judgment criteria for the F2 generation results are as follows:
8.1 In each test tube, if there are no wild-type male flies with red round eyes in more than 20 offspring (male and female), it is positive, which is a lethal mutation. If there are more than 2 wild-type male flies with red round eyes, it is negative. 8.2 In each test tube, if there are less than 20 offspring or only one wild-type male fly, it is necessary to observe the F2 generation. 8.3 Infertility: Only male and female parents are left without offspring. 80
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