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HG3617—1999
This standard is formulated based on the relevant materials such as the Bacillus thuringiensis enterprise standards formulated in the past in my country and combined with the actual situation in my country. This standard makes specific requirements and provisions for the requirements, test methods, sampling, packaging, transportation, etc. of Bacillus thuringiensis wettable powder, thus providing a unified technical basis for the production of Bacillus thuringiensis. This standard was proposed by the Technical Supervision Department of the former Ministry of Chemical Industry of the People's Republic of China. This standard is under the jurisdiction of the Shenyang Chemical Research Institute of the Ministry of Chemical Industry. The main drafting unit of this standard: Department of Applied Chemistry, China Agricultural University. The participating drafting units of this standard: Hubei Biopesticide Engineering Research and Development Center, Jinan Kerbel Bioengineering Co., Ltd. The main drafters of this standard: Liu Fengmao, Wang Bingmei, Qian Chuanfan, Zhong Liansheng, Zhao Xinxin, Wang Qiwen. 1166
Chemical Industry Standard of the People's Republic of China
Bacillus thuringiensis wettable powder
Bacillus thuringiensis wettable powderHG3617--1999
Bacillus thuringiensis (Bt) is the most widely used microbial insecticide. Its main insecticide component is the toxin protein in the parasporal crystal, among which the relative molecular mass of the protein toxic to Lepidoptera is 130000.1 Scope
This standard specifies the requirements, test methods, and marking, labeling, packaging, storage and transportation of Bacillus thuringiensis wettable powder. This standard applies to Bacillus thuringiensis wettable powder made from Bacillus thuringiensis technical and adjuvants, which is mainly used to control Lepidoptera pests.
2 Referenced Standards
The provisions contained in the following standards constitute the provisions of this standard through reference in this standard. All versions are valid when this standard is published. All standards will be revised. All parties using this standard should explore the possibility of using the latest version of the following standards. GB/T1250-1989 Method for expressing and determining limit values GB/T1600-1979 (1989) Method for determining moisture content of pesticides GB/T1601-1993 Method for determining pH value of pesticides GB/T1604-1995 Acceptance rules for commercial pesticides GB/T1605-1979 (1989) Method for sampling commercial pesticides GB3796-1983 General rules for pesticide packaging || tt||GB/T5451-1984 Method for determination of wettability of pesticide wettable powders GB/T14825—1993 Method for determination of suspension rate of pesticide wettable powders GB/T16150—1995 Method for determination of fineness of pesticide powders and wettable powders HG3616--1999 Bacillus thuringiensis powder
3 Requirements
3.1 Appearance: off-white to brown loose powder, without lumps. 3.2 Bacillus thuringiensis wettable powder shall meet the requirements of Table 1. Table 1 Control indicators of Bacillus thuringiensis wettable powder Item
Toxin protein, %
Toxicity titer ([Px IU /mgJ[Ha IU/mgJ)pH value
Fineness (75μm), %
Approved by the State Administration of Petroleum and Chemical Industry on June 16, 199932000IU/mg
16 000 IU/mg
8000IU/mg
Implemented on June 1, 2000
Suspension rate (active ingredient), %
Wetting time, min
Water·%
HG 3617—1999
Table 1 (complete)
32 000 IU/mg
16 000 IU/mg
Note: Px and Ha are the abbreviations of Plutella xylostella and Heliothis armigera, respectively. 4 Test methods
Unless otherwise specified, all reagents used in this method are analytically pure and all solutions are aqueous solutions. 4.1 Sampling
8 000 IU/mg
According to Chapter 5 "Sampling of Powders and Wettable Powders" of GB/T1605-1979 (1989), the sampling packages are determined by the random number table method. The final sampling volume should not be less than 100g. 4.2 Identification test
When doubts arise in the detection of product quality by bioassay, the following method can be used for identification. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method is used to determine whether the relative molecular mass of the effective toxin protein is 130,000, and at the same time, the toxin protein content is determined to meet the requirements of the indicators in 3.2. 4.3 Determination of toxin protein content
The toxin protein content can be determined by SDS-PAGE scanning method and SDS-PAGE elution colorimetry. The two methods have similar precision and accuracy. The former is designated as the arbitration method because of its higher degree of automation. 4.3.1 SDS-PAGE scanning method (arbitration method) 4.3.1.1 Method summary
Treat the spore-associated crystals of thuringia strychnifolia wettable powder with an alkaline solution to degrade them into toxin proteins. Then, by SDS-PAGE, the toxin proteins are separated from other impurities according to the difference in the relative molecular mass of the proteins. Then, the protein band area is scanned with a thin layer scanner or an electrophoresis image scanner for quantitative determination.
4.3.1.2 Instruments and equipment
Electrophoresis instrument.
Sandwich-type vertical electrophoresis tank (1.5mm concave groove rubber mold frame), gel plate area 145mmX100mm (1.5mm, 12-hole sample tank mold).
High-speed thin-layer chromatography scanner or electrophoresis image scanner. Centrifuge: 10 000r/min.
Analytical balance: accurate to 0.0001g.
4.3.1.3 Reagents and solutions
Ammonium persulfate (AP).
Sodium dodecyl sulfate (SDS).
Tetramethylethylenediamine (TEMED).
Sodium hydroxide.
30% acrylamide: weigh 30g acrylamide and 0.8g methylenebisacrylamide (formerly known as methylenebisacrylamide), dissolve in 100mL distilled water, filter, and store in a dark place at 4℃ for later use. 1mol/L, pH8.8 tris(hydroxymethylaminomethane)-HCl buffer: weigh 30.25g tris(hydroxymethylaminomethane) and dissolve in distilled water, adjust to pH8.8 with concentrated hydrochloric acid, and dilute to 250mL with distilled water. 1 mol/L, pH 6.8 trihydroxyaminomethane-HCl buffer: weigh 12.10 g trihydroxyaminomethane and dissolve it in distilled water, adjust to pH 6.8 with concentrated 1168
HG 3617—1999
hydrochloric acid, and dilute to 100 mL with distilled water. Electrode buffer: weigh 3.03 g trihydroxyaminomethane, 14.42 g glycine, 1 g sodium dodecyl sulfate, dissolve in distilled water and dilute to 1000 mL.
3× sample diluent: 18.75 mL 1 mol/L, pH 6.8 trihydroxyaminomethane-HCl, 6 g sodium dodecyl sulfate, 30 mL glycerol, 15 mL mercaptoethanol, a little bromophenol blue, dilute to 100 mL with distilled water. Staining solution: weigh 1g of Coomassie Brilliant Blue (CBB) R-250, add 450mL of methanol, 100mL of glacial acetic acid, and 450mL of distilled water, dissolve and filter before use.
Decolorizing solution: measure 100mL of methanol, 35mL of glacial acetic acid and dilute to 1000mL with distilled water. Rinsing solution: measure 30mL of anhydrous ethanol, 10mL of glacial acetic acid, and 60mL of distilled water, mix well before use. Toxin protein standard sample: original powder with a toxin protein (relative molecular weight of 130000) content of 9.3%. 4.3.1.4 Sample treatment
Weigh 20mg of standard sample and sample each (accurate to 0.1mg), transfer to 5mL centrifuge, and add 2mL of water to fully suspend. Then add 0.45mL of 0.55mol/L sodium hydroxide solution (so that the final concentration of sodium hydroxide solution is 0.1mol/L), let it stand for about 5 minutes, then add 1.30mL of 3× sample diluent to make the final volume 3.75mL, boil in 100℃ distilled water for 6 minutes, centrifuge (2000r/min) for 10 minutes, and then take the supernatant to prepare for electrophoresis. 4.3.1.5SDS-PAGE separation of toxin protein
a) Preparation of 8%~10% polyacrylamide gel
Use discontinuous buffer system, gel preparation method see Appendix A of HG3616---1999 (suggested appendix). b) Sample loading
Take the above standard sample to dissolve the supernatant, and load 6, 8, 10, 12, and 14μL (the toxin protein content is about 3-7μg) in the loading well of the polyacrylamide gel respectively as the standard curve, and then take a certain volume of the supernatant of the sample solution (the toxin protein content is about 5μg), add it to the loading well, inject the electrode buffer, and turn on the power. c) Electrophoresis
The initial voltage of electrophoresis is controlled at about 100V. After the sample enters the separation gel, increase the voltage to 120V and continue electrophoresis. When the indicator front reaches about 1cm from the bottom, stop electrophoresis, take out the gel plate, and soak it in 7.5% (volume percentage) acetic acid for 30min. d) Staining
Remove the separation gel and stain it with Coomassie Brilliant Blue (CBB) R-250 staining solution overnight. e) Decolorization
Pour off the staining solution, wash the gel with rinse solution first, then add decolorizing solution, heat at 37°C to decolorize it, and change the decolorizing solution several times until the background is clear.
4.3.1.6 Determination
After decolorization of the gel plate, the 130,000 protein band can be clearly seen. Scan the band with a high-speed thin layer chromatography scanner or electrophoresis image scanner at a scanning wavelength of 600nm.
The percentage of toxin protein in the sample (X) is calculated according to formula (1). mVz×100
Where: m1 -- the amount of toxin protein in the sample obtained from the standard curve, ug, m -- the mass of the sample in 2 mL of diluent, mg; Vi -- the final volume of the sample, mL (3.75 mL), V2 -- the volume of the sample injected into the gel loading hole, μL. 4.3.1.7 Allowable difference
The arithmetic mean is taken as the determination result. The relative deviation of the results of two parallel determinations is less than or equal to 8%. 4.3.2 SDS-PAGE-elution colorimetric method
4.3.2.1 Method summary
HG 3617 -- 1999
After treating the spore-associated crystals of Bacillus thuringiensis WP with an alkaline solution to degrade them into toxin protein, the toxin protein is separated from other impurities by SDS-PAGE according to the difference in the relative molecular mass of the protein, and then the gel is cut, eluted, and the absorbance is determined. 4.3.2.2 Instruments and equipment
Spectrophotometer.
Others are the same as 4.3.1.2.
4.3.2.3 Reagents and solutions
Pyridine.
Others are the same as 4.3.1.3.
4.3.2.4 Sample treatment
Same as 4.3.1.4.
4.3.2.5 SDS-PAGE separation of toxin protein
a) Prepare 8%~10% polyacrylamide gel as in 4.3.1.5a).
b) Sample loading
Take the supernatant of the above standard sample solution and load 15, 20, 30, 40, and 50uL (the toxin protein content is about 7.5~~25μg) into the loading wells respectively. As the standard curve, take a fixed volume of the supernatant of the sample solution (the toxin protein content is about 15μg), add it to the sample well, inject the electrode buffer, and turn on the power. c) Electrophoresis
Same as 4.3.1.5c).
d) Staining
Same as 4.3.1.5d).
e) Decolorization
Same as 4.3.1.5e).
4.3.2.6 Determination
Use a scalpel to scrape the test zone, put it in a glass test tube, add 3.0mL of 25% pyridine (volume percentage), shake at 37°C to elute the Cox Brilliant Blue (CBB) R-250 adsorbed by the toxin protein, and after equilibrium, use a spectrophotometer with 25% pyridine as a reference to measure the absorbance of the solution at 605nm, and calculate the toxin protein content using formula (1). 4.3.2.7 Allowable difference
Take the arithmetic mean as the test result. The relative deviation of the results of two parallel determinations is less than or equal to 8%. 4.4 Determination of toxicity
Perform according to Appendix B of HG 3616--1999 (Standard Appendix). 4.5 Determination of pH value
Perform according to GB/T1601.
4.6 Determination of fineness
Perform according to 2.2 of GB/T16150--1995. 4.7 Determination of suspension rate
4.7.1 Operation steps
Weigh 200.0 mg of sample (accurate to 0.1 mg), put it in a triangular flask containing glass beads, add 100 mL of standard hard water, and shake it left and right by hand for 60 times. Transfer all the obtained suspension to a 250 mL stoppered volumetric cylinder and dilute it to 250 mL with standard hard water. According to 3.1 of GB/T14825--93.
4.7.2 Calculation
The suspension rate (Y) is calculated according to formula (2):
HG 3617--1999
Y= 111.1(C
Wherein: C—the toxicity titer of the sample taken to prepare the suspension, IU; Q
The toxicity titer of the 25mL suspension left at the bottom of the measuring cylinder, IU. 4.7.3 Allowable difference
The difference between the results of the two repeated measurements should not exceed 10%. 4.8 Determination of wetting time
According to GB/T5451.
4.9 Determination of moisture
According to the "azeotropic distillation method" in GB/T1600--1979 (1989). 5 Inspection rules
Should comply with the relevant provisions of GB/T1604. Limit values shall be handled in accordance with GB/T1250. 6 Marking, labeling, packaging, storage and transportation
6.1 Product packaging shall comply with the provisions of GB3796 and shall indicate the standard code used. 6.2 Wettable powder products are mainly packaged in sealed plastic bags. 6.3 When storing, avoid direct sunlight and pressure, and place in a cool and dry place. 6.4 When transporting, handle with care to prevent damage. (2)
6.5 Guarantee period: Under normal storage and transportation conditions, the quality guarantee period of wettable powder is two years from the date of production. The toxicity titer and toxin protein content of the product shall not be less than 3.2 indicators when it leaves the factory. Within two years, the toxicity titer and toxin protein content of the product shall not be less than 70% of the 3.2 indicators. 11711.5a).
b) Sample loading
Take the supernatant of the above standard solution and load 15, 20, 30, 40, and 50uL (the toxin protein content is about 7.5~~25μg) in the loading wells respectively. As the standard curve, take a fixed volume of the supernatant of the sample solution (the toxin protein content is about 15μg), add it to the loading well, inject the electrode buffer, and turn on the power. c) Electrophoresis
Same as 4.3.1.5c).
d) Staining
Same as 4.3.1.5d).
e) Decolorization
Same as 4.3.1.5e).
4.3.2.6 Determination
Use a scalpel to scrape the test zone, place it in a glass test tube, add 3.0mL of 25% pyridine (volume percentage), and shake at 37°C to elute the Cox Brilliant Blue (CBB) R-250 adsorbed by the toxin protein. After equilibrium, use a spectrophotometer to measure the absorbance of the solution at 605nm with 25% pyridine as a reference, and calculate the toxin protein content using formula (1). 4.3.2.7 Allowable difference
Take its arithmetic mean as the determination result. The relative deviation of the results of two parallel determinations is less than or equal to 8%. 4.4 Determination of toxicity
Perform according to Appendix B of HG 3616--1999 (Appendix to the standard). 4.5 Determination of pH value
Perform according to GB/T1601.
4.6 Determination of fineness
Perform the determination according to 2.2 of GB/T16150--1995. 4.7 Determination of suspension rate
4.7.1 Operation steps
Weigh 200.0 mg of sample (accurate to 0.1 mg), put it into a triangular flask containing glass beads, add 100 mL of standard hard water, and shake it left and right by hand for 60 times. Transfer all the obtained suspension to a 250 mL stoppered measuring cylinder and dilute it to 250 mL with standard hard water. Perform according to 3.1 of GB/T14825--93.
4.7.2 Calculation
The suspension rate (Y) is calculated according to formula (2):
HG 3617-—1999
Y= 111.1(C
Wherein: C—the toxicity titer of the sample taken to prepare the suspension, IU; Q
the toxicity titer of the 25mL suspension left at the bottom of the measuring cylinder, IU. 4.7.3 Allowable difference
The difference between the results of two repeated measurements should not exceed 10%. 4.8 Determination of wetting time
According to GB/T5451.
4.9 Determination of moisture
According to the "azeotropic distillation method" in GB/T1600--1979 (1989). 5 Inspection rules
Should comply with the relevant provisions of GB/T1604. Limit values shall be handled in accordance with GB/T1250. 6 Marking, labeling, packaging, storage and transportation
6.1 Product packaging shall comply with the provisions of GB3796 and shall indicate the standard code used. 6.2 Wettable powder products are mainly packaged in sealed plastic bags. 6.3 When storing, avoid direct sunlight and pressure, and place in a cool and dry place. 6.4 When transporting, handle with care to prevent damage. (2)
6.5 Guarantee period: Under normal storage and transportation conditions, the quality guarantee period of wettable powder is two years from the date of production. The toxicity titer and toxin protein content of the product shall not be less than 3.2 indicators when it leaves the factory. Within two years, the toxicity titer and toxin protein content of the product shall not be less than 70% of the 3.2 indicators. 11711.5a).
b) Sample loading
Take the supernatant of the above standard solution and load 15, 20, 30, 40, and 50uL (the toxin protein content is about 7.5~~25μg) in the loading wells respectively. As the standard curve, take a fixed volume of the supernatant of the sample solution (the toxin protein content is about 15μg), add it to the loading well, inject the electrode buffer, and turn on the power. c) Electrophoresis
Same as 4.3.1.5c).
d) Staining
Same as 4.3.1.5d).
e) Decolorization
Same as 4.3.1.5e).
4.3.2.6 Determination
Use a scalpel to scrape the test zone, place it in a glass test tube, add 3.0mL of 25% pyridine (volume percentage), and shake at 37°C to elute the Cox Brilliant Blue (CBB) R-250 adsorbed by the toxin protein. After equilibrium, use a spectrophotometer to measure the absorbance of the solution at 605nm with 25% pyridine as a reference, and calculate the toxin protein content using formula (1). 4.3.2.7 Allowable difference
Take its arithmetic mean as the determination result. The relative deviation of the results of two parallel determinations is less than or equal to 8%. 4.4 Determination of toxicitybZxz.net
Perform according to Appendix B of HG 3616--1999 (Appendix to the standard). 4.5 Determination of pH value
Perform according to GB/T1601.
4.6 Determination of fineness
Perform the determination according to 2.2 of GB/T16150--1995. 4.7 Determination of suspension rate
4.7.1 Operation steps
Weigh 200.0 mg of sample (accurate to 0.1 mg), put it into a triangular flask containing glass beads, add 100 mL of standard hard water, and shake it left and right by hand for 60 times. Transfer all the obtained suspension to a 250 mL stoppered measuring cylinder and dilute it to 250 mL with standard hard water. Perform according to 3.1 of GB/T14825--93.
4.7.2 Calculation
The suspension rate (Y) is calculated according to formula (2):
HG 3617-—1999
Y= 111.1(C
Wherein: C—the toxicity titer of the sample taken to prepare the suspension, IU; Q
the toxicity titer of the 25mL suspension left at the bottom of the measuring cylinder, IU. 4.7.3 Allowable difference
The difference between the results of two repeated measurements should not exceed 10%. 4.8 Determination of wetting time
According to GB/T5451.
4.9 Determination of moisture
According to the "azeotropic distillation method" in GB/T1600--1979 (1989). 5 Inspection rules
Should comply with the relevant provisions of GB/T1604. Limit values shall be handled in accordance with GB/T1250. 6 Marking, labeling, packaging, storage and transportation
6.1 Product packaging shall comply with the provisions of GB3796 and shall indicate the standard code used. 6.2 Wettable powder products are mainly packaged in sealed plastic bags. 6.3 When storing, avoid direct sunlight and pressure, and place in a cool and dry place. 6.4 When transporting, handle with care to prevent damage. (2)
6.5 Guarantee period: Under normal storage and transportation conditions, the quality guarantee period of wettable powder is two years from the date of production. The toxicity titer and toxin protein content of the product shall not be less than 3.2 indicators when it leaves the factory. Within two years, the toxicity titer and toxin protein content of the product shall not be less than 70% of the 3.2 indicators. 1171
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