Some standard content:
HG3618—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 suspension, 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 Kaimei, Qian Chuanfan, Zhong Liansheng, Zhao Xinxin, Wang Qiwen. 1172
Chemical Industry Standard of the People's Republic of China
Bacillus thuringiensis suspension concentrate
Bacillus thuringiensis suspension concentrate HG 3618--1999
Bacillus thuringiensis (Bt) is currently 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 130,000.1 Scope
This standard specifies the requirements, test methods, and marking, labeling, packaging, storage and transportation of Bacillus thuringiensis suspension concentrate. This standard applies to Bacillus thuringiensis suspension concentrate 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 are subject to revision. Parties using this standard should explore the possibility of using the latest versions of the following standards. GB/T1250—1989 Methods for expressing and determining limit values GB/T1601—1993 Methods for determining pH value of pesticides GB/T1604-1995 Rules for acceptance of commercial pesticides GB/T1605—1979 (1989) Methods for sampling commercial pesticides GB3796—1983 General rules for pesticide packaging
GB/T148251993 Methods for determining the suspension rate of pesticide wettable powders GB/T16150—1995 Methods for determining the fineness of pesticide powders and wettable powders HG3617—1999 Bacillus thuringiensis raw powder
3 Requirements
3.1 Appearance: Brownish yellow to brown suspension liquid. 3.2 Bacillus thuringiensis suspension concentrates shall also meet the requirements of Table 1. Table 1 Indicators of Bacillus thuringiensis suspension control items
Toxin protein, %
Toxicity titer ([Px IU /μLJ[Ha IU/μLJ)pH range
Particle size (150 μm), %
Suspension rate (active ingredient), %
8 000 IU/μL
4 000IU/μL
Note: Px and Ha are the abbreviations for Plutella xylostella and Heliothis armigera, respectively. Approved by the State Administration of Petroleum and Chemical Industry on June 16, 1999, 2 000 IU/μL
Implemented on June 1, 2000
4 Test Method
HG 3618-1999
Unless otherwise specified, all reagents used in this method are analytically pure, and all solutions are aqueous solutions. 4.1 Sampling
According to Chapter 4 "Sampling of Emulsions and Liquid States" of GB/T1605-1979 (1989), the number of packages to be sampled is determined by the random number table method, and the final sampling volume should be no less than 250 mL. 4.2 Identification Test
When doubts arise about the quality of the product detected by the bioassay method, 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
The spore-associated crystals of Bacillus thuringiensis suspension are treated with 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 relative molecular weight 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 vertical electrophoresis tank (1.5mm concave groove rubber mold frame), gel plate area 145mm×100mm (1.5mm, 12-hole sample tank mold).
High-speed thin-layer chromatography scanner or electrophoresis image scanner. Centrifuge: 10000r/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. 1 mol/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 make up to 250mL with distilled water. 1 mol/L, pH 6.8 tris(hydroxymethylaminomethane)-HCl buffer: weigh 12.10 g of tris(hydroxymethylaminomethane) and dissolve it in distilled water, adjust to pH 6.8 with concentrated hydrochloric acid, and dilute to 100 mL with distilled water. Electrode buffer: weigh 3.03 g of tris(hydroxymethylaminomethane), 14.42 g of glycine, and 1 g of sodium dodecyl sulfate, dissolve in distilled water and dilute to 1000 mL.
3× sample diluent: 18.75 mL of 1 mol/L, pH 6.8 tris(hydroxymethylaminomethane)-HCl, 6 g of sodium dodecyl sulfate, 30 mL of glycerol, 15 mL of mercaptoethanol, a little bromophenol blue, dilute to 100 mL with distilled water. Staining solution: weigh 1 g of Coomassie Brilliant Blue (CBB) R-250, add 450 mL of methanol, 100 mL of glacial acetic acid, and 450 mL of distilled water, dissolve and filter before use.
Decolorizing solution: Measure 100mL of methanol and 35mL of glacial acetic acid, and dilute to 1000mL with distilled water. 1174
HG 3618-- 1999
Rinsing solution: Measure 30mL of anhydrous ethanol, 10mL of glacial acetic acid, and 60mL of distilled water, mix well and use. Toxin protein standard sample: original powder with a toxin protein content of 9.3% (relative molecular weight of 130000). 4.3.1.4 Sample treatment
Weigh 20mg of the standard sample (accurate to 0.1mg), transfer it to a 5mL centrifuge tube, and add 2mL of water to fully suspend it. Measure 10mL of the sample to be tested, weigh it accurately, and dilute to 100mL with distilled water. After fully shaking, take 2mL of the diluent and transfer it to a 5mL centrifuge tube.
Add 0.45mL of 0.55mol/L sodium hydroxide solution to the above 2ml sample solution (so that the final concentration of the sodium hydroxide solution is 0.1mol/L), let it stand for about 5min, then add 1.30mL of 3× sample diluent to make the final volume 3.75mL, boil in 100℃ distilled water for 6min, centrifuge (2000r/min) for 10min, and take the supernatant to prepare for electrophoresis. 4.3.1.5 SDS-PAGE separation of toxin protein
a) Preparation of 8%~10% polyacrylamide gel
Use a discontinuous buffer system. For the gel preparation method, see Appendix A of HG3616-1999 (suggested appendix). b) Sample loading
Take the supernatant of the above standard sample solution and load 6.8, 10, 12, 14 μL (the toxin protein content is about 3-7 μg) as the standard curve, and then take a certain volume of the supernatant of the sample solution (the toxin protein content is about 5 μg), add it to the sample 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 1 cm from the bottom, stop electrophoresis, take out the gel plate, and soak it in 7.5% (volume percentage) acetic acid for 30 minutes. d) Staining
Remove the separation gel part 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 it at 37°C to decolorize it, and change the decolorizing solution several times until the background is clear. bzxZ.net
4.3.1.6 Determination
After the gel plate is decolorized, the 130000 protein band can be clearly seen. Scan the band with a high-speed thin layer chromatography scanner or electrophoresis image scanner, and the scanning wavelength is 600nm.
The percentage content (X) of toxin protein in the sample is calculated according to formula (1). X=mV
Wherein: ml—the amount of toxin protein in the sample obtained from the standard curve, μg; m22 The mass of the sample in mL diluent, mg; V,--the final volume of the sample, mL (3.75mL); V2——the volume of the sample injected into the gel loading hole, uL. 4.3.1.7 Allowable difference
Take the arithmetic mean 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
+*（1）
Treat the parasporal crystals of Bacillus thuringiensis suspension with alkaline solution to degrade them into toxin proteins. Then, separate the toxin proteins from other impurities by SDS-PAGE according to the difference in relative molecular weight of the proteins. Then cut the gel, elute and measure the absorbance. 4.3.2.2 Apparatus
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
Same as 4.3.1.5a).
b) Sample loading
HG 3618-1999
Take the supernatant of the above standard solution and load 15, 20, 30, 40, and 50μL (the toxin protein content is about 7.5~25ug) in the loading wells respectively as the standard curve, then take a certain volume of the supernatant of the sample solution (the toxin protein content is about 15μg), add it to the loading wells, 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 Coomassie 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 suspension rate
4.6.1 Determination steps
After the sample is spread evenly, take 5.00mL (accurate to 0.01mL) and put it in a 100mL triangular glass bottle. Add 100mL of standard hard water and shake it left and right by hand for 50 times. Transfer all the obtained suspension to a 250mL stoppered measuring cylinder and dilute it to 250mL with standard hard water. Perform according to 3.1 of GB/T14825-1993.
4.6.2 Calculation
The suspension rate (Y) is calculated according to formula (2):
Y = 111.1(C - Q)
Wherein: C—-the toxicity of the sample taken to prepare the suspension, IU; Q——the toxicity of the 25mL suspension left at the bottom of the measuring tube, IU. 4.6.3 Allowable difference
The difference between the results of two repeated determinations shall not exceed 10%. 4.7 Determination of fineness
Pipette 20.00mL of sample (accurate to 0.01mL) and proceed according to 2.2 of GB/T16150-1995. 1176
(2)
5 Inspection rules
HG3618-1999
Should comply with the relevant provisions of GB/T1604. The limit value shall be handled according to GB/T1250. 6 Marking, labeling, packaging, storage and transportation
6.1 Product packaging should comply with the provisions of GB3796, and the standard number used should be indicated. 6.2 Suspension concentrate products are mainly packaged in 1L plastic bottles with inner and outer covers to prevent leakage. 6.3 When storing, strictly prevent sunlight and pressure, and place in a cool and dry place. 6.4 During transportation, be careful to handle with care to prevent damage. 6.5 Guarantee period: Under normal transportation conditions, the quality assurance period of the suspension concentrate is one and a half years from the date of production. The toxicity titer and protein content of this product are not lower than the 3.2 index when leaving the factory. Within one and a half years, the toxicity titer and protein content are not lower than 60% of the 3.2 index. 11776 Determination
Use a scalpel to scrape off the test zone, place it in a glass test tube, add 3.0 mL of 25% pyridine (volume percentage), and shake at 37°C to elute the Coomassie Brilliant Blue (CBB) R-250 adsorbed by the toxin protein. After equilibrium, use a spectrophotometer to measure the absorbance of the solution at 605 nm 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 suspension rate
4.6.1 Determination steps
After the sample is spread evenly, take 5.00mL (accurate to 0.01mL) and put it in a 100mL triangular glass bottle. Add 100mL of standard hard water and shake it left and right by hand for 50 times. Transfer the obtained suspension to a 250mL stoppered volumetric cylinder and dilute it to 250mL with standard hard water. Follow 3.1 of GB/T14825-1993.
4.6.2 Calculation
The suspension rate (Y) is calculated according to formula (2):
Y = 111.1(C - Q)
Where: 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 volumetric cylinder, IU. 4.6.3 Allowable difference
The difference between the results of two repeated determinations shall not exceed 10%. 4.7 Determination of fineness
Pipette 20.00mL of sample (accurate to 0.01mL) and proceed according to 2.2 of GB/T16150-1995. 1176
(2)
5 Inspection rules
HG3618-1999
Should comply with the relevant provisions of GB/T1604. The limit value 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 number used. 6.2 Suspension concentrate products are mainly packaged in 1L plastic bottles with inner and outer covers and no leakage. 6.3 When storing, strictly prevent sunlight and pressure, and place in a cool and dry place. 6.4 When transporting, be careful to handle with care to prevent damage. 6.5 Warranty period: Under normal operating conditions, the quality assurance period of the suspension is one and a half years from the date of production. The toxicity titer and protein content of this product shall not be lower than the 3.2 index when it leaves the factory. Within one and a half years, the toxicity titer and protein content shall not be lower than 60% of the 3.2 index.6 Determination
Use a scalpel to scrape off the test zone, place it in a glass test tube, add 3.0 mL of 25% pyridine (volume percentage), and shake at 37°C to elute the Coomassie Brilliant Blue (CBB) R-250 adsorbed by the toxin protein. After equilibrium, use a spectrophotometer to measure the absorbance of the solution at 605 nm 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 suspension rate
4.6.1 Determination steps
After the sample is spread evenly, take 5.00mL (accurate to 0.01mL) and put it in a 100mL triangular glass bottle. Add 100mL of standard hard water and shake it left and right by hand for 50 times. Transfer the obtained suspension to a 250mL stoppered volumetric cylinder and dilute it to 250mL with standard hard water. Follow 3.1 of GB/T14825-1993.
4.6.2 Calculation
The suspension rate (Y) is calculated according to formula (2):
Y = 111.1(C - Q)
Where: 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 volumetric cylinder, IU. 4.6.3 Allowable difference
The difference between the results of two repeated determinations shall not exceed 10%. 4.7 Determination of fineness
Pipette 20.00mL of sample (accurate to 0.01mL) and proceed according to 2.2 of GB/T16150-1995. 1176
(2)
5 Inspection rules
HG3618-1999
Should comply with the relevant provisions of GB/T1604. The limit value 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 number used. 6.2 Suspension concentrate products are mainly packaged in 1L plastic bottles with inner and outer covers and no leakage. 6.3 When storing, strictly prevent sunlight and pressure, and place in a cool and dry place. 6.4 When transporting, be careful to handle with care to prevent damage. 6.5 Guarantee period: Under normal operating conditions, the quality guarantee period of the suspension is one and a half years from the date of production. The toxicity titer and protein content of this product shall not be lower than the 3.2 index when it leaves the factory. Within one and a half years, the toxicity titer and protein content shall not be lower than 60% of the 3.2 index.
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