HG 3616-1999 English PDF (HG3616-1999)

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HG 3616-1999: [HG/T 3616-1999] Bacillus thuringiensis technical
HG 3616-1999
HG
CHEMICAL INDUSTRY STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
Bacillus thuringiensis technical
APPROVED ON: JUNE 16, 1999
IMPLEMENTED ON: JUNE 01, 2000
Approved by: National Bureau of Petroleum and Chemical Industry
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative references ... 4
3 Requirements ... 4
4 Test methods ... 5
5 Inspection rules ... 10
6 Marks, labels, packaging, storage and transportation... 10
Annex A (informative) Preparation of electrophoretic gel ... 11
Annex B (standard) Determination of virulence potency ... 13
Bacillus thuringiensis technical
Bacillus thuringiensis (B. t.) is the most widely used microbial insecticide. Its main
insecticidal component is the toxin protein in the parasporal crystal. The relative
molecular mass of the protein that is toxic to Lepidoptera is 130000.
1 Scope
This Standard specifies the requirements, test methods, marks, labels, packaging,
storage and transportation of Bacillus thuringiensis raw powder.
This Standard applies to the original powder of Bacillus thuringiensis for the control of
Lepidoptera pests.
2 Normative references
The following standards contain provisions which, through reference in this Standard,
constitute provisions of this Standard. At the time of publication, the editions indicated
are valid. All standards are subject to revision. The parties who are using this Standard
shall explore the possibility of using the latest version of the following standards.
GB/T 1250-1989, Rules for expression and judgement of limiting values
GB/T 1600-1979 (1989), Determination of the moisture content in pesticides
GB/T 1601-1993, Determination method of pH value for pesticides
GB/T 1604-1995, Commodity pesticide regulations for acceptance
GB/T 1605-1979 (1989), Sampling method for commercial pesticides
GB 3796-1983, General rule for packing of pesticides
GB/T 16150-1995, Sieve test for dustable and wettable powders of pesticides
3 Requirements
3.1 Appearance: Off-white to tan powder.
3.2 The raw powder of Bacillus thuringiensis shall meet the requirements in Table 1.
4.3.1.2 Instruments, equipment
Electrophoresis.
Sandwich type vertical electrophoresis tank (1.5mm concave rubber frame with groove),
gel plate area 145mm × 100mm (1.5mm, 12-well sample tank mold).
High-speed thin-layer tomography scanner or electrophoretic 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 of acrylamide, 0.8g of methylene bisacrylamide (formerly
known as: methylene bisacrylamide). Dissolve in 100mL of distilled water. Filter. Store
at 4°C in a dark place for future use.
1mol/L, pH8.8 trishydroxymethylaminomethane-HCl buffer solution: Weigh 30.25g of
trishydroxymethylaminomethane and dissolve in distilled water. Adjust to pH 8.8 with
concentrated hydrochloric acid. Use distilled water to set volume constant to 250mL.
1mol/L, pH6.8 trishydroxymethylaminomethane-HCl buffer solution: Weigh 12.10g of
trishydroxymethylaminomethane and dissolve in distilled water. Adjust to pH 6.8 with
concentrated hydrochloric acid. Use distilled water to set volume constant to 100mL.
Electrode buffer solution: Weigh 3.03g of trishydroxymethylaminomethane, 14.42g of
glycine, 1g of sodium lauryl sulfate. Use water to dissolve and set volume constant to
1000mL.
3X sample diluent: 18.75mL of 1mol/L, pH6.8 trishydroxymethylaminomethane-HCl,
6g of sodium lauryl sulfate, 30mL of glycerin, 15mL of mercaptoethanol, a little
bromophenol blue. Use distilled water to set volume constant to 100mL.
Staining solution: Weigh 1g of Coomassie Brilliant Blue (CBB) R-250. Add 450mL of
methanol, 100mL of glacial acetic acid, 450mL of distilled water. Dissolve and filter
for future use.
Decolorizing solution: Measure 100mL of methanol, 35mL of glacial acetic acid. Use
distilled water to set volume constant to 1000mL.
Rinse solution: Measure 30mL of absolute ethanol, 10mL of glacial acetic acid, and
60mL of distilled water. Mix well before use.
Toxin protein standard sample: Raw powder of which toxin protein (relative molecular
mass is 130000) content is 9.3%.
4.3.1.4 Specimen processing
Weigh 20mg of standard sample and specimen (accurate to 0.1mg) respectively.
Transfer to a 5mL centrifuge tube. 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). Place for about 5min. Then add 1.30mL of 3X sample
diluent. Make the final volume to 3.75mL. Boil for 6min in 100°C boiling water.
Centrifuge (at 2000 r/min) for 10min. Take the supernatant, so as to prepare samples
for electrophoresis.
4.3.1.5 SDS-PAGE separation of toxin proteins
a) Prepare 8%~10% polyacrylamide gel
Use the discontinuous buffer system. For the gel making method, see Annex A
(informative).
b) Sample loading
Take the supernatant of the above-mentioned standard sample solution. Load 6,
8, 10, 12, and 14μL of samples into the sample wells of the polyacrylamide gel
respectively (toxin protein content is about 3~7μg), as the standard curve. Then
take a certain volume of the supernatant of the specimen solution (toxin protein
content is about 5μg). Add to the sample well. After injecting the electrode buffer,
turn on the power.
c) Electrophoresis
The initial voltage of electrophoresis is controlled at about 100V. After the
specimen enters the separation gel, increase the voltage to 120V and continue
electrophoresis. Stop electrophoresis when the front of the indicator reaches about
1cm from the bottom. Take out the gel plate. Soak in 7.5% (volume percent) acetic
acid for 30min.
d) Dyeing
Remove the separation gel. Stain overnight with Coomassie brilliant blue (CBB)
R-250 staining solution.
e) Decolorizing
Pyridine.
Others are the same as 4.3.1.3.
4.3.2.4 Specimen processing
Same as 4.3.1.4.
4.3.2.5 SDS-PAGE separation of toxin proteins
a) Prepare 8%~10% polyacrylamide gel
Same as 4.3.1.5a).
b) Sample loading
Take the supernatant of the above standard solution. Load 15, 20, 30, 40, 50μL of
samples into the sample wells respectively (toxin protein content is about
7.5~25μg), as the standard curve. Then take a certain volume of the supernatant
of the specimen solution (toxin protein content is about 15μg). Add to the sample
well. After injecting the electrode buffer, turn on the power.
c) Electrophoresis
Same as 4.3.1.5c).
d) Dyeing
Same as 4.3.1.5d).
e) Decolorizing
Same as 4.3.1.5e).
4.3.2.6 Determination
Scrape off the zone to be tested with a scalpel. Put in a glass test tube. Then add 3.0mL
of 25% pyridine (volume fraction). Shake at 37°C to elute the Cox Brilliant Blue (CBB)
R-250 adsorbed by the toxin protein. After equilibration use a spectrophotometer. With
reference to 25% pyridine, at 605nm, measure the absorbance of the solution. The toxin
protein content is calculated by formula (1).
4.3.2.7 Allowable difference
Take the arithmetic mean value as the determination result. The relative deviation of
the two parallel determination results is less than or equal to 8%.
4.4 Determination of virulence potency
Annex A
(informative)
Preparation of electrophoretic gel
A1 Plate preparation