GB/T 38475-2020.Detection of mycotoxins in pigment-Rapid quantitative method of colloidal gold strip test.
1 Scope
GB/T 38475 specifies the method for detecting biotoxins in natural pigment products by colloidal gold rapid quantitative method.
GB/T 38475 is applicable to the rapid quantitative detection of aflatoxin B, citrinin, zearalenone and vomitoxin in natural pigment products prepared by extraction and fermentation of natural products.
2 Normative references
The following documents are indispensable for the application of this document. For all dated references, only the dated version applies to this document. For all undated references, the latest version (including all amendments) applies to this document.
GB/T 5491 Sampling and splitting methods for grain and oil inspection
GB/T 6682 Specifications and test methods for water in analytical laboratories
3 Principle
The biological toxins in the sample extract compete with the specific antibodies labeled with colloidal gold particles in the test reagent strip during the chromatography process and produce a color reaction. The color depth is related to the content of biological toxins in the sample. The color depth of the test line and the quality control line on the test strip is verified by a reader, and the content of biological toxins in the sample is automatically calculated based on the color depth and the built-in curve of the reader.
4 Reagents or materials.
Unless otherwise specified, all reagents used are analytically pure.
4.1 Water
GB/T 6682, secondary water.
4.2 Aflatoxin B
Purity ≥98%.
4.3 Citrinin
Purity ≥98%.
4.4 Zearalenone
Purity ≥ 98%.
4.5 Deoxytoxin
Purity ≥ 98%.
4.6 Benzene + acetonitrile mixture
Measure 98.0 mL of benzene, add 2.0 mL of acetonitrile, and mix well.
4.7 Dilution buffer
Weigh 2.422 8 g of tris(hydroxymethyl)aminomethane, dissolve it in water, adjust pH=8.0; add 9.0 g of sodium chloride and 5 mL of Triton X-100, and make up to 1 000 mL with water; or use commercially available similar products.
4.8 70% (volume fraction) methanol solution extract
Take 70.0 mL of methanol and add 30.0 mL of water and mix well.
4.9 PBS buffer solution
Weigh 8.0 g sodium chloride, 1.2 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate, and 0.2 g potassium chloride, dissolve in water, adjust pH to 7.0, and dilute to 1 000 mL with water.
4.10 0.1% (volume fraction) Tween 20-PBS solution
Pipette 1 mL Tween 20 and dilute to 1 000 mL with PBS buffer solution.
This standard specifies the method for rapid quantitative detection of biological toxins in natural pigment products using colloidal gold. This standard is applicable to the rapid quantitative detection of aflatoxin B1, citrinin, zearalenone, and vomitoxin in natural pigment products prepared by extraction and fermentation of natural products.

ICS07.080
National Standard of the People's Republic of China
GB/T38475—2020
Detection of mycotoxins in pigment-Rapid quantitative method of colloidal gold strip test2020-11-19 Issued
State Administration for Market Regulation
National Standardization Administration
Issued
2020-11-19 Implementation
Foreword
This standard was drafted in accordance with the rules given in GB/T1.1-2009. This standard was proposed by the China National Institute of Standardization and is under the jurisdiction of GB/T38475-2020
Drafting units of this standard: China University of Metrology, China National Institute of Standardization, Jiangnan University, Beijing Sambo Technology Co., Ltd. Main drafters of this standard: Cui Haifeng, Ye Zihong, Ma Aijin, Liu Liqiang, Zhang Mingzhou, Yu Xiaoping, Xu Yipeng, Shen Juxuping, Zhang Yafen, Kuang Hua, Hao Shuai.
1 Scope
Detection of biological toxins in pigments
Colloidal gold rapid quantitative method
This standard specifies the method for detecting biological toxins in natural pigment products using colloidal gold rapid quantitative method. GB/T38475—2020
This standard applies to the rapid quantitative detection of aflatoxin Bi, citrinin, zearalenone and vomitoxin in natural pigment products prepared by extraction and fermentation of natural products. Normative references
The following documents are indispensable for the application of this document. For all dated references, only the dated version applies to this document. For all undated references, the latest version (including all amendments) applies to this document. GB/T5491 Sampling and splitting methods for grain and oilseed inspection GB/T6682
Specifications and test methods for water in analytical laboratories
The biological toxins in the sample extract compete with the specific antibodies labeled with colloidal gold particles in the test reagent strip during the chromatography process and produce a color reaction. The depth of color is related to the content of biological toxins in the sample. Use a reader to verify the color depth of the test line and the quality control line on the test strip, and automatically calculate the content of biological toxins in the sample based on the color depth and the built-in curve of the reader. Reagents or materials
Unless otherwise specified, all reagents used are analytically pure. 4.1 Water
GB/T6682, secondary water. bZxz.net
2 Aflatoxin B,
Purity ≥98%.
Citrin
Purity ≥98%.
Zerallarenone
Purity ≥98%.
5 Deoxytoxin
Purity ≥98%.
GB/T38475—2020
Benzene-decanoyl cyanide mixture
Measure 98.0mL of benzene, add 2.0mL of acetonitrile, and mix well. 4.7
Dilution buffer
Weigh 2.4228g tris(hydroxymethyl)aminomethane, dissolve in water, adjust pH=8.0; add 9.0g sodium chloride and 5mL TritonX-100, dilute to 1000mL with water; or use commercially available similar products. 370% (volume fraction) methanol solution extract 4.8
Take 70.0mL of methanol and add 30.0mL of water, mix PBS buffer solution
Weigh 8.0g sodium chloride, 1.2g disodium hydrogen phosphate, 0.2g potassium dihydrogen phosphate, 0.2g potassium chloride, dissolve in water, adjust pH to 7.0, and dilute to 1000mL with water.
4.100.1% (volume fraction) Tween 20-PBS solution Take 1mL Tween 20 and dilute to 1000mL with PBS buffer solution. 4.110.1% (volume fraction) phosphoric acid solution Accurately transfer 1mL of phosphoric acid and add water to make up to 1000mL. 4.120.1mg/L biotoxin standard stock solution Accurately weigh a certain amount of biotoxin standard, dissolve it with benzotriazole (98:2) solution, make up to 10mL in a volumetric flask, prepare 0.1mg/L biotoxin standard stock solution, store it at 4℃ for standby use, and can be stored for 1 year. 5 Instruments and equipment
5.1 Balance: accuracy 0.01g and 0.01mg. 2 Pulverizer: speed not less than 3000r/min. 5.2
3 Centrifuge: speed not less than 3000×g. 5.3
5.4 Jiang
Vortex oscillator.
Reader: can measure and display the test results of colloidal gold immunochromatographic test strips 6 Colloidal gold immunochromatographic test strips: performance requirements are shown in Appendix A. 5.6
Micropipette.
Immunoaffinity column: column volume specification 1mL or 3mL, column capacity not less than 200ng, or equivalent column sample preparation
Sample sampling
Perform according to GB/T5491.
6.2 Sample preparation
6.2.1 Solid sample
GB/T38475—2020
Weigh 500g of representative solid or sample on a balance, of which 300g is crushed with a grinder until all pass through a 20-mesh sieve, mix well, and seal and freeze in two portions. Take a sample for storage and accurately weigh 2.0g of the sample into a 10mL stoppered conical bottle, add 20.0mL of 70% methanol solution extract, seal, vortex oscillator for 2min3min, let stand and filter with filter paper, or take 1.5mL of the mixture into a centrifuge tube and centrifuge for 1min. Take 100μL of the filtrate or supernatant after centrifugation and put it in another centrifuge tube, add 900μL to the dilution buffer, and mix thoroughly for testing. If the sample is a special material, the diluted mixed solution needs to be filtered with a syringe filter, which is the test solution. 6.2.2 Liquid sample
The liquid sample to be tested is treated with a homogenizer for no less than 2 minutes. After being fully mixed, use a micropipette to pipette 1.0mL into two 2.0mL centrifuge tubes, one of which is sealed and frozen for storage, and the other is used for testing. Pipette 200μL of the centrifuged liquid sample from the centrifuge tube to be tested into another centrifuge tube, add 1.8mL of dilution buffer, and mix thoroughly for testing. 6.2.3 Purification
Connect the immunoaffinity column to a 10mL glass syringe, accurately transfer 10.0mL of the above sample filtrate to the immunoaffinity column, and pass it through the affinity column at a flow rate of 1 drop/s to 2 drops/s; add 10mL of 0.1% Tween 20-PBS solution, and elute the column at a flow rate of 1 drop/s to 2 drops/s until air enters the affinity column, and discard all the effluent. Accurately add 1.0mL of eluent for elution, and the elution flow rate is 1 drop/s to 2 drops/s. Collect all the eluent in a centrifuge tube for later use. Note: The purification process is only for pigment products such as red yeast rice that have serious background interference with the colloidal gold reaction results. 7 Determination
7.1 After taking out the test strips stored at low temperature, place them at room temperature for 20min to 30min. Colloidal gold reagent strip: Immerse the test strip in a centrifuge tube containing 300μL~500μL of the test solution and incubate at room temperature for 5min7.2
7.3 Colloidal gold test card: Add 60μL~80uL of the test solution to the test well and incubate at room temperature for 5min. It is advisable to use the reader of the colloidal gold test strip or test card manufacturer. 7.4
8 Result analysis
8.1 After the reaction is completed, take out the colloidal gold immunochromatographic test strip or test card to observe the color development of the C line (quality control line) and the T line (test line). If the C line does not appear, or the C line appears but is diffuse or severely uneven, or the C line appears but the T line is diffuse or severely uneven, it is considered an invalid test.
8.2 Adjust the parameters of the reader, fix the colloidal gold test strip or test card, and detect the target toxin. The measurement needs to be completed within 2 minutes and repeated 3 times. The reader automatically calculates and displays the content of the corresponding biological toxin in the sample according to the built-in fitting curve, in micrograms per kilogram (ug/kg) or micrograms per liter (μg/L). 8.3 Within the detection range of the corresponding biological toxin, based on the negative control sample, set different concentration gradients for adding blank samples, among which the concentration range of aflatoxin B is 2ug/kg128ug/kg; the concentration range of citrinin is 40μg/kg~640μg/kg; the concentration range of vomitoxin is 300μg/kg14400ug/kg; the concentration range of zearalenone is 40ug/kg~1920μg/kg. Based on the standard sample concentration value and the integrated area detection data representing the T line intensity optical density value, the standard curve is established using the four-parameter logistic fitting algorithm! The fitting degree R2 value should be between 0.99 or above.
GB/T38475—2020
Repeatability
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 20% of the arithmetic mean. 10 Others
The detection limits of this method are 2.0μg/kg for aflatoxin B, 40ug/kg for citrinin, 60μg/kg for zearalenone, and 500μg/kg for vomitoxin.
A.1 Accuracy
Appendix A
(Normative Appendix)
Performance requirements for colloidal gold rapid test strips GB/T38475—2020
Use physical standard samples at concentrations of 2, 5, and 8 times the minimum detection limit, and measure each concentration level no less than 6 times. Calculate the deviation between the test results of the test strip and the physical standard sample. The deviations of the three concentration levels should be controlled between -20% and +20%. A.2 Precision
Use a physical standard sample with a concentration level 5 times the minimum detection limit, measure no less than 6 times, and calculate the coefficient of variation within the test strip batch. The coefficient of variation is ≤20%.
A.3 Detection limit
Measure 20 negative samples, calculate the average value plus 3 times the standard deviation, and the result should be less than or equal to the product sensitivity indication value a
A.4 Inter-batch stability
Use a physical standard sample with a concentration level of about 10ug/kg for testing, which shall not be less than 6 batches, and each batch shall be measured no less than 2 times. Take the average value of the intra-batch measurement, and calculate the coefficient of variation between the test strip batches. The coefficient of variation should be less than or equal to 20%. A.5
Specificity
Select a negative test sample, add other biotoxins that may cross-react with the target detection biotoxin, and except for the target detection biotoxin added sample with a positive test result, the test results of the other added samples are all negative5
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