1 Scope
This standard specifies the examination method of clostridium botulinum and botulinum toxin in foods.
This standard is applicable to the examination of clostridium botulinum and botulinum toxin in foods.
2 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
2.1 Refrigerator: 2℃~5℃, -20℃.
2.2 Balance: with sensibility of 0.1g.
2.3 Aseptic surgical scissors, tweezers and reagent scoop.
2.4 Homogenizer or aseptic mortar.
2.5 Centrifuge: 3 000r/min, 14 000r/min.
2.6 Anaerobic culture apparatus.
2.7 Constant temperature incubator: 35℃±1℃, 28℃±1℃.
2.8 Thermostatic water bath: 37℃±1℃, 60℃±1℃, 80℃±1℃.
2.9 Microscope: 10x~100x.
2.10 PCR instrument.
2.11 Electrophoresis apparatus or capillary electrophoresis apparatus.
2.12 Gel imaging system or ultraviolet detector.
2.13 Nucleic acid protein analyzer or ultraviolet spectrophotometer.
2.14 Adjustable micropipettor: 0.2μL~2μL, 2μL~20μL, 20μL~200μL, 100μL~1 000μL.
2.15 Aseptic pipette: 1.0mL, 10.0mL, 25.0mL.
2.16 Aseptic conical flask: 100mL.
2.17 Culture dish: 90mm in diameter.
2.18 Centrifugal tube: 50mL, 1.5mL.
2.19 PCR reaction tube.
2.20 Aseptic injector: 1.0mL.
2.21 Mice: 15g~20g, the KM or ICR mice with same strain shall be adopted for each batch of test.
3 Media and Reagents
Unless otherwise specified, the reagents for PCR test are analytically pure or in conformity with biochemical reagent criteria, and the water shall meet the requirements of Grade I water in GB/T 6682.
3.1 Cooked meat medium: see A.1.
3.2 Trypsin peptone glucose yeast extract broth (TPGYT): see A.2.
3.3 Yolk agar medium: see A.3.
3.4 Gelatin phosphate buffer solution: see A.4.
3.5 Gram stain solution: see A.5.
3.6 10% trypsin solution: see A.6.
3.7 Phosphate buffer solution (PBS): see A.7.
3.8 1mol/L sodium hydroxide solution.
3.9 1mol/L hydrochloric acid solution.
3.10 Botulinum toxin diagnostic serum.
3.11 Absolute ethanol and 95% ethanol.
3.12 10mg/mL lysozyme solution.
3.13 10mg/mL proteinase K solution.
3.14 3mol/L sodium acetate solution (pH 5.2)
3.15 TE buffer solution.
3.16 Primer: synthetized according to the sequence in Table 1 and prepared with ultrapure water in concentration of 10μmol/L right before use.
3.17 10×PCR buffer solution.
3.18 25mmol/L MgCl2.
3.19 dNTPs: dATP, dTTP, dCTP, dGTP.
3.20 Taq enzyme.
3.21 Agarose: electrophoresis-grade.
3.22 Ethidium bromide or Goldview.
3.23 5×TBE buffer solution.
3.24 6×loading buffer solution.
3.25 DNA molecular weight standard.
4 Examination Procedures
See Figure 1 for the examination procedures of clostridium botulinum and botulinum toxin.
Figure 1 Examination Procedures of Clostridium Botulinum and Botulinum Toxin
5 Operation Steps
5.1 Preparation of sample
5.1.1 Preservation of sample
The to-be-examined sample shall be stored in a refrigerator at 2℃~5℃.
5.1.2 Solid and semi-solid foods
For solid or semi-solid food with little free liquid, weigh 25g of sample by aseptic technique, and put it into aseptic homogenizing bag or aseptic mortar, cut up the blocky food by aseptic technique. Add 25mL of gelatin phosphate buffer solution to solid food containing more water and 50mL to such foods containing less water as milk powder and beef jerky, soak for 30min, flap with slap-type homogenizer for 2min or grind with aseptic pestle to prepare homogeneous sample solution, and then collect for standby.
5.1.3 Liquid foods
Shake up the liquid food and weigh 25mL by aseptic technique for examination.
5.1.4 Treatment of residual samples
Place the residual sample after sampling in refrigerator at 2℃~5℃ until the examination result report is sent out, carry out innocent treatment according to infectious waste, the sample detected to be positive shall be subjected to innocent treatment by pressure steam sterilization.
5.2 Detection of Botulinum toxin
5.2.1 Preparation of toxin solution
Take 40mL of homogeneous sample solution or 25mL of homogeneous liquid sample into centrifugal tube, centrifuge at 3 000r/min for 10min~20min, collect supernatant and divide it into two portions and put them into aseptic test tubes respectively, of which one is used for toxin detection directly and the other for toxin detection after pancreatin treatment. Reserve the bottom sediments and liquid abt. 12mL for liquid sample, and then re-suspend it for preparation of sediment suspension for standby.
Pancreatin treatment: adjust the pH value of supernatant to 6.2 with 1mol/L of sodium hydroxide or 1mol/L of hydrochloric acid, mix well the supernatant and 10% pancreatin (with vitality of 1:250) according to the ratio of 9:1, hatch it at 37℃ for 60min, during the period, slightly shake the reaction liquid occasionally.
5.2.2 Detection test
Take 0.5mL of centrifugal supernatant and pancreatin-treated supernatant by No. 5 pinhead injector to carry out intraperitoneal injection for each one of three mice, observe and record the intoxication performance of mice within 48h. Typical toxicity symptom of botulinum toxin occurs within 24h in most cases; generally, mice get ill and then die within 6h with major manifestations as piloerection, weak and limp limbs, dyspnea, presenting bellows-type respiration, depression of waist and abdomen just like peak waist, and death mainly caused by respiratory failure, which may be preliminarily judged as botulinum toxin intoxication. If mice get ill or die after 24h, observe their symptoms carefully, and if necessary, re-conduct the test with concentrated supernatant to eliminate botulinum toxin intoxication. If mice suddenly die (within 30min) giving rise to unobvious symptoms, the toxin supernatant shall be properly diluted and then re-conduct the test.
Note: toxin detection test of animal shall comply with the provisions of “National Food Safety Standard—Good Laboratory Practice for Food Edures forToxicology” (GB 15193.2).
5.2.3 Confirmatory test
For the positive toxin test of supernatant or (and) pancreatin treatment supernatant, take three portions of 0.5mL of corresponding test solution, therein, add equivalent amount of polytypic hybrid botulinum toxin diagnostic serum into the first portion, mix well and incubate at 37℃ for 30min, add equivalent amount of gelatin phosphate buffer solution into the second portion, mix well and boil for 10min, and add equivalent amount of gelatin phosphate buffer solution into the third portion and mix well. Respectively take 0.5mL of three portions of mixed solution to carry out intraperitoneal injection for each one of two mice, and observe the intoxication and death condition of mice within 96h.
Result judgment: if the mice injected with the first and second portions of mixed solution survive while those injected with the third portion of solution get ill and die as well as the peculiar symptom of botulinum toxin intoxication occurs, then it is judged that botulinum toxin is detected in the sample.
5.2.4 Virulence determination (optional)
Take the test solution verified to be positive and use gelatin phosphate buffer solution to prepare diluent of certain dilution ratio such as 10 times, 50 times, 100 times and 500 times. Respectively take 0.5mL of the test solution to carry out intraperitoneal injection for each one of two mice, observe and record their morbidity and death until 96h, calculate the minimum lethal dose (MLD/mL or MLD/g), and evaluate the botulinum toxin virulence of sample; MLD is equal to the highest dilution ratio of entire death of mice multiplied by the dilution ratio of sample test solution. For example, if the test solution obtained by diluting the supernatant prepared with double diluted sample by 100 times makes all mice die, while the 500-fold dilution solution makes mice survive, the virulence of sample is 200MLD/g.
5.2.5 Type test (optional)
According to the virulence determination result, use gelatin phosphate buffer solution to dilute the supernatant to 10MLDmL~1000MLD/mL as the type test solution, respectively mix with equal amount of various monotypic botulinum toxin diagnostic serum (generally, domestic diagnostic serum refers to freeze-dried serum which is dissolved with 1mL of normal saline), incubate at 37℃ for 30min, respectively take 0.5mL to carry out intraperitoneal injection for each one of two mice, observe and record their morbidity and death until 96h. Meanwhile, use gelatin phosphate buffer solution to replace diagnostic serum and mix with equivalent amount of test solution to serve as the mice test control.
Result judgment: animals in a certain monotypic diagnostic serum group don't get ill and survive normally, while animals in control group and other monotypic diagnostic serum groups get ill and die, so it is judged that the botulinum toxin included in the sample is the type of botulinum toxin.
Note: the positive toxin detection test or confirmatory test of sample supernatant without pancreatin activation treatment, the pancreatin activation treatment may be omitted for virulence determination and type test.
5.3 Examination of clostridium botulinum
5.3.1 Enrichment cultivation and detection test
5.3.1.1 Take out four cooked meat media and two TPGY broth tubes, boil by water isolation for 10min~15min, eliminate dissolved oxygen, rapidly cool it and never shake, slowly add 1mL of pancreatin solution into TPGY broth tube to the broth below liquid paraffin liquid level to prepare TPGYT.
5.3.1.2 Pipet 2mL of homogeneous sample solution or centrifugal sediment suspension in the process of toxin preparation to inoculate to cooked meat medium, inoculate four pcs. for each sample, directly place two of which at 35℃±1℃ for anaerobic cultivation for 5 d, place the other two at 80℃ for insulation for 10min and then place it at 35℃±1℃ for anaerobic cultivation for 5d; inoculate two TPGYT broth tubes by the same method at 28℃±1℃ for anaerobic cultivation for 5d.
Contents
Foreword I
1 Scope
2 Apparatus and Materials
3 Media and Reagents
4 Examination Procedures
5 Operation Steps
6 Result Report
Appendix A Media and Reagents
1 Scope
This standard specifies the examination method of clostridium botulinum and botulinum toxin in foods.
This standard is applicable to the examination of clostridium botulinum and botulinum toxin in foods.
2 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
2.1 Refrigerator: 2℃~5℃, -20℃.
2.2 Balance: with sensibility of 0.1g.
2.3 Aseptic surgical scissors, tweezers and reagent scoop.
2.4 Homogenizer or aseptic mortar.
2.5 Centrifuge: 3 000r/min, 14 000r/min.
2.6 Anaerobic culture apparatus.
2.7 Constant temperature incubator: 35℃±1℃, 28℃±1℃.
2.8 Thermostatic water bath: 37℃±1℃, 60℃±1℃, 80℃±1℃.
2.9 Microscope: 10x~100x.
2.10 PCR instrument.
2.11 Electrophoresis apparatus or capillary electrophoresis apparatus.
2.12 Gel imaging system or ultraviolet detector.
2.13 Nucleic acid protein analyzer or ultraviolet spectrophotometer.
2.14 Adjustable micropipettor: 0.2μL~2μL, 2μL~20μL, 20μL~200μL, 100μL~1 000μL.
2.15 Aseptic pipette: 1.0mL, 10.0mL, 25.0mL.
2.16 Aseptic conical flask: 100mL.
2.17 Culture dish: 90mm in diameter.
2.18 Centrifugal tube: 50mL, 1.5mL.
2.19 PCR reaction tube.
2.20 Aseptic injector: 1.0mL.
2.21 Mice: 15g~20g, the KM or ICR mice with same strain shall be adopted for each batch of test.
3 Media and Reagents
Unless otherwise specified, the reagents for PCR test are analytically pure or in conformity with biochemical reagent criteria, and the water shall meet the requirements of Grade I water in GB/T 6682.
3.1 Cooked meat medium: see A.1.
3.2 Trypsin peptone glucose yeast extract broth (TPGYT): see A.2.
3.3 Yolk agar medium: see A.3.
3.4 Gelatin phosphate buffer solution: see A.4.
3.5 Gram stain solution: see A.5.
3.6 10% trypsin solution: see A.6.
3.7 Phosphate buffer solution (PBS): see A.7.
3.8 1mol/L sodium hydroxide solution.
3.9 1mol/L hydrochloric acid solution.
3.10 Botulinum toxin diagnostic serum.
3.11 Absolute ethanol and 95% ethanol.
3.12 10mg/mL lysozyme solution.
3.13 10mg/mL proteinase K solution.
3.14 3mol/L sodium acetate solution (pH 5.2)
3.15 TE buffer solution.
3.16 Primer: synthetized according to the sequence in Table 1 and prepared with ultrapure water in concentration of 10μmol/L right before use.
3.17 10×PCR buffer solution.
3.18 25mmol/L MgCl2.
3.19 dNTPs: dATP, dTTP, dCTP, dGTP.
3.20 Taq enzyme.
3.21 Agarose: electrophoresis-grade.
3.22 Ethidium bromide or Goldview.
3.23 5×TBE buffer solution.
3.24 6×loading buffer solution.
3.25 DNA molecular weight standard.
4 Examination Procedures
See Figure 1 for the examination procedures of clostridium botulinum and botulinum toxin.
Figure 1 Examination Procedures of Clostridium Botulinum and Botulinum Toxin
5 Operation Steps
5.1 Preparation of sample
5.1.1 Preservation of sample
The to-be-examined sample shall be stored in a refrigerator at 2℃~5℃.
5.1.2 Solid and semi-solid foods
For solid or semi-solid food with little free liquid, weigh 25g of sample by aseptic technique, and put it into aseptic homogenizing bag or aseptic mortar, cut up the blocky food by aseptic technique. Add 25mL of gelatin phosphate buffer solution to solid food containing more water and 50mL to such foods containing less water as milk powder and beef jerky, soak for 30min, flap with slap-type homogenizer for 2min or grind with aseptic pestle to prepare homogeneous sample solution, and then collect for standby.
5.1.3 Liquid foods
Shake up the liquid food and weigh 25mL by aseptic technique for examination.
5.1.4 Treatment of residual samples
Place the residual sample after sampling in refrigerator at 2℃~5℃ until the examination result report is sent out, carry out innocent treatment according to infectious waste, the sample detected to be positive shall be subjected to innocent treatment by pressure steam sterilization.
5.2 Detection of Botulinum toxin
5.2.1 Preparation of toxin solution
Take 40mL of homogeneous sample solution or 25mL of homogeneous liquid sample into centrifugal tube, centrifuge at 3 000r/min for 10min~20min, collect supernatant and divide it into two portions and put them into aseptic test tubes respectively, of which one is used for toxin detection directly and the other for toxin detection after pancreatin treatment. Reserve the bottom sediments and liquid abt. 12mL for liquid sample, and then re-suspend it for preparation of sediment suspension for standby.
Pancreatin treatment: adjust the pH value of supernatant to 6.2 with 1mol/L of sodium hydroxide or 1mol/L of hydrochloric acid, mix well the supernatant and 10% pancreatin (with vitality of 1:250) according to the ratio of 9:1, hatch it at 37℃ for 60min, during the period, slightly shake the reaction liquid occasionally.
5.2.2 Detection test
Take 0.5mL of centrifugal supernatant and pancreatin-treated supernatant by No. 5 pinhead injector to carry out intraperitoneal injection for each one of three mice, observe and record the intoxication performance of mice within 48h. Typical toxicity symptom of botulinum toxin occurs within 24h in most cases; generally, mice get ill and then die within 6h with major manifestations as piloerection, weak and limp limbs, dyspnea, presenting bellows-type respiration, depression of waist and abdomen just like peak waist, and death mainly caused by respiratory failure, which may be preliminarily judged as botulinum toxin intoxication. If mice get ill or die after 24h, observe their symptoms carefully, and if necessary, re-conduct the test with concentrated supernatant to eliminate botulinum toxin intoxication. If mice suddenly die (within 30min) giving rise to unobvious symptoms, the toxin supernatant shall be properly diluted and then re-conduct the test.
Note: toxin detection test of animal shall comply with the provisions of “National Food Safety Standard—Good Laboratory Practice for Food Edures forToxicology” (GB 15193.2).
5.2.3 Confirmatory test
For the positive toxin test of supernatant or (and) pancreatin treatment supernatant, take three portions of 0.5mL of corresponding test solution, therein, add equivalent amount of polytypic hybrid botulinum toxin diagnostic serum into the first portion, mix well and incubate at 37℃ for 30min, add equivalent amount of gelatin phosphate buffer solution into the second portion, mix well and boil for 10min, and add equivalent amount of gelatin phosphate buffer solution into the third portion and mix well. Respectively take 0.5mL of three portions of mixed solution to carry out intraperitoneal injection for each one of two mice, and observe the intoxication and death condition of mice within 96h.
Result judgment: if the mice injected with the first and second portions of mixed solution survive while those injected with the third portion of solution get ill and die as well as the peculiar symptom of botulinum toxin intoxication occurs, then it is judged that botulinum toxin is detected in the sample.
5.2.4 Virulence determination (optional)
Take the test solution verified to be positive and use gelatin phosphate buffer solution to prepare diluent of certain dilution ratio such as 10 times, 50 times, 100 times and 500 times. Respectively take 0.5mL of the test solution to carry out intraperitoneal injection for each one of two mice, observe and record their morbidity and death until 96h, calculate the minimum lethal dose (MLD/mL or MLD/g), and evaluate the botulinum toxin virulence of sample; MLD is equal to the highest dilution ratio of entire death of mice multiplied by the dilution ratio of sample test solution. For example, if the test solution obtained by diluting the supernatant prepared with double diluted sample by 100 times makes all mice die, while the 500-fold dilution solution makes mice survive, the virulence of sample is 200MLD/g.
5.2.5 Type test (optional)
According to the virulence determination result, use gelatin phosphate buffer solution to dilute the supernatant to 10MLDmL~1000MLD/mL as the type test solution, respectively mix with equal amount of various monotypic botulinum toxin diagnostic serum (generally, domestic diagnostic serum refers to freeze-dried serum which is dissolved with 1mL of normal saline), incubate at 37℃ for 30min, respectively take 0.5mL to carry out intraperitoneal injection for each one of two mice, observe and record their morbidity and death until 96h. Meanwhile, use gelatin phosphate buffer solution to replace diagnostic serum and mix with equivalent amount of test solution to serve as the mice test control.
Result judgment: animals in a certain monotypic diagnostic serum group don't get ill and survive normally, while animals in control group and other monotypic diagnostic serum groups get ill and die, so it is judged that the botulinum toxin included in the sample is the type of botulinum toxin.
Note: the positive toxin detection test or confirmatory test of sample supernatant without pancreatin activation treatment, the pancreatin activation treatment may be omitted for virulence determination and type test.
5.3 Examination of clostridium botulinum
5.3.1 Enrichment cultivation and detection test
5.3.1.1 Take out four cooked meat media and two TPGY broth tubes, boil by water isolation for 10min~15min, eliminate dissolved oxygen, rapidly cool it and never shake, slowly add 1mL of pancreatin solution into TPGY broth tube to the broth below liquid paraffin liquid level to prepare TPGYT.
5.3.1.2 Pipet 2mL of homogeneous sample solution or centrifugal sediment suspension in the process of toxin preparation to inoculate to cooked meat medium, inoculate four pcs. for each sample, directly place two of which at 35℃±1℃ for anaerobic cultivation for 5 d, place the other two at 80℃ for insulation for 10min and then place it at 35℃±1℃ for anaerobic cultivation for 5d; inoculate two TPGYT broth tubes by the same method at 28℃±1℃ for anaerobic cultivation for 5d.
Contents of GB 4789.12-2016
Contents
Foreword I
1 Scope
2 Apparatus and Materials
3 Media and Reagents
4 Examination Procedures
5 Operation Steps
6 Result Report
Appendix A Media and Reagents
