LS/T 6108-2014 Grain and Oil Inspection Rapid Determination of Aflatoxin B1 in Cereals by Immunochromatographic Method LS/T6108-2014 |tt||Standard compression package decompression password: www.bzxz.net
This standard specifies the principle, reagents and materials, instruments and equipment, sample preparation, sample determination, result determination and positive sample confirmation of enzyme-labeled immunochromatographic method and colloidal gold immunochromatographic method for aflatoxin B1 in cereals.
This standard was drafted in accordance with the rules given in GB/T1.1-2009. This standard is
under the jurisdiction of the National Technical Committee for Standardization of Cereals and Oils (SAC/TC270). This standard
was drafted by: Hubei Provincial Cereals, Oils and Food Quality Monitoring Station, Standard and Quality Center of the State Administration of Grain, Beijing Cereals, Oils and Food Inspection Institute, Chongqing Cereals and Oils Quality Supervision and Inspection Station, Guangxi Zhuang Autonomous Region Cereals and Oils Quality Supervision and Inspection Station.
The main drafters of this standard are: Xiong Ning, Liu Jian, Yang Weimin, Guo Jian, Liu Yongying, Zou Yong, Peng Chao, Liu Li, Liu Yong, Ni Shanshan, Wu Lili.
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.
GB2761 National Food Safety Standard Limits of Mycotoxins in Food
GB5491 Sampling and Splitting Methods for Grain and Oilseed Inspection
GB/T6682 Specifications and Test Methods for Water Used in Analytical Laboratories

ICS67.060
Registration No.: 44929—2014
Grain Industry Standard of the People's Republic of China
LS/T6108—2014
Grain and oils inspection
Rapid test of aflatoxin B, in cereals-Immunochromatography method
Inspection of grain and oils-Rapid test of aflatoxin B, in cereal-Immunochromatography method
Published on April 28, 2014
State Administration of Grain
Implementation on June 1, 2014
This standard was drafted according to the rules given in GB/T1.1-2009. This standard is under the jurisdiction of the National Technical Committee for Standardization of Cereals and Oils (SAC/TC270) LS/T6108—2014
The drafting units of this standard are: Hubei Cereals and Oils Food Quality Monitoring Station, Standard and Quality Center of the State Grain Administration, Beijing Cereals and Oils Food Inspection Institute, Chongqing Cereals and Oils Quality Supervision and Inspection Station, Guangxi Zhuang Autonomous Region Cereals and Oils Quality Supervision and Inspection Station. The main drafters of this standard are: Xiong Ning, Liu Jian, Yang Weimin, Guo Jian, Liu Yongying, Zou Yong, Peng Chao, Liu Li, Liu Yong, Ni Yanna, Wu Lili. 1 Scope
Cereals and Oils Inspection
Rapid Determination of Aflatoxin B in Cereals by Immunochromatography
LS/T6108-—2014
This standard specifies the principle, reagents and materials, instruments and equipment, sample preparation, sample determination, and result judgment of enzyme-labeled immunochromatography and colloidal gold immunochromatography for the determination of aflatoxin B in cereals. This standard applies to the rapid screening of aflatoxin B in rice, brown rice, corn and other cereals. 2 Normative references
The following documents are essential for the application of this document. For dated references, only the dated version applies to this document. For undated references, the latest version (including all amendments) applies to this document GB2761 National Food Safety Standard Limits of Mycotoxins in Food GB5491 Sample Support and Sample Separation Method for Grain and Oil Inspection GB/T6682 Specifications and Test Methods for Water in Analytical Laboratories 3 Enzyme-labeled Immunochromatography
3.1 Principle
This method is based on specific antigen-antibody reaction and immunochromatography technology, with enzyme as carrier and double antibody sandwich reaction principle. The sample is extracted with methanol solution, and the extract is filtered or centrifuged. After release, it is added to the test card. Aflatoxin B in the sample will bind to the antibody on the solid phase carrier and then to the enzyme-labeled antibody on the test strip, making the test strip color. The content of aflatoxin B in the sample can be determined by whether the test strip is colored and how long the color development time is. 3.2 Reagents and Materials
Unless otherwise specified, all reagents used are analytically pure, and the laboratory water should comply with the first-level water in GB/T6682. 3.2.1 Aflatoxin B ELISA Card\: The performance of the test card should meet the requirements of Appendix A. Before using test cards of different brands and batches, the performance of the test card should be determined in accordance with Appendix A. 3.2.2 70% methanol solution: Take 70mL methanol and add 30mL water to mix. 3.2.3 Disodium hydrogen phosphate (Na,HPO,).
3.2.4 Sodium fluoride (NaCI).
3.2.5 Potassium chloride (KCI).
3.2.6 Concentrated hydrochloric acid (HCI).
1) The aflatoxin B ELISA card produced by Germany's Bayer is a rapid test card that is currently widely used. This information is provided only for the convenience of the users of this standard and does not represent the approval of this product by this standard. Any test card that meets the requirements of Appendix A of this standard can be used. bzxz.net
LS/T6108-—2014
3.2.7PBS buffer: Weigh 1.42g disodium hydrogen phosphate, 8g sodium chloride, and 0.2g potassium chloride respectively, add 800mL water and stir to dissolve, add concentrated hydrochloric acid to adjust the pH to 7.4, and dilute to 1L with water. 3.3
Instruments and equipment
In addition to conventional laboratory instruments and equipment, the following instruments and equipment should be noted. 3.3.1 Balance: graduation value 0.01g, range 210g. 3.3.2 Pulverizer.
3.3.3 Centrifuge: speed 4000r/min. 3.3.4 Oscillator.
3.3.5 Timer.
3.3.6 Centrifuge tube.
3.4 ​​Sample preparation
Sample support and sample splitting: Follow GB5491.
3.4.2 Sample crushing: Crush the sample to be tested until it passes through a 20-mesh sieve and mix thoroughly. 3.4.3 Preparation of test solution: Accurately weigh 10.0 g of the crushed sample (3.4.2) into a centrifuge tube and add 20 mL of 70% methanol solution (3.2.2). Oscillate and extract for 3 min 5 min, let stand for 3 min, filter or centrifuge to obtain the supernatant. Take 100 μL of PBS buffer (3.2.7) at room temperature and mix it with 50 μL of the supernatant. This solution is the test solution. Note: The sample should be fully mixed before weighing to ensure the representativeness of the sample to be tested: 3.5 Sample determination
Take 100 μL of the test solution (3.4.3) and add it to the spot hole of the test card (3.2.1), start timing, and read the results three times within 16 min.
Note: The quality control color band should appear on the test card every time it is tested, and the quality control color band will appear within 2 minutes after adding the sample. 3.6 Result determination
3.6.1 Invalid
If the quality control color band does not show color, this test card is invalid and a new test card must be used for retesting. 3.6.2 Positive and negative samples
If the quality control color band shows color and the test color band shows color at different times, it means that the content of aflatoxin B in the sample is different. If the test color band shows color after 4 minutes, the content of aflatoxin B in the sample is about 20uμg/kg: if the test color band shows color after 8 minutes, the content of aflatoxin B in the sample is about 10μg/kg: if the test color band shows color after 16 minutes, the content of aflatoxin B in the sample is about 4μg/kg. The positive and negative of the sample is determined according to the limit standards for aflatoxin B in various grains specified in GB2761. When it is necessary to confirm the positive samples screened by this method, the method specified in GB2761 should be used. 4 Colloidal gold immunochromatography
4.1 Principle
This method is based on specific antigen-antibody reaction and immunochromatography technology, with colloidal gold as the carrier and the reaction principle of the competitive method. The sample is extracted with ethyl acetate, and the extract is filtered or centrifuged, dried, diluted, and added to the test card. Aflatoxin B in the sample will bind to the specific antibody labeled with 2
LS/T6108-2014
colloidal gold, so that the detection color band does not show color. The content of aflatoxin B in the sample is determined by detecting whether the color band is colored.
4.2 Reagents and materials
Unless otherwise specified, all reagents used are analytically pure, and the laboratory water should comply with the first-level water in GB/T6682. 4.2.1 Aflatoxin B, colloidal gold test card 2": The performance of the test card should meet the requirements of Appendix A. Before using different brands and batches, the performance of the test card should be measured in accordance with Appendix A. 4.2.2 Ethyl acetate.
4.2.3 Disodium hydrogen phosphate (NaHPO,).
4.2.4 Sodium chloride (NaCI).
4.2.5 Potassium chloride (KCI).
4.2.6 Concentrated hydrochloric acid (HCI).
4.2.7 PBS buffer: Same as 3.2.7.
Instruments and equipment
In addition to conventional laboratory instruments and equipment, the following instruments and equipment should be noted. 4.3.1 Balance, graduation value 0.01g, range 210g. 4.3.2 Pulverizer.
4.3.3 Centrifuge Centrifuge: speed 4000r/min. 4.3.4 Oscillator.
4.3.5 Timer.
4.3.6 Electric hair dryer: 500W~1000W.
4.3.7 Centrifuge tube.
4.3.8 Glass beaker.
Sample preparation
Sampling and sub-sampling: same as 3.4.1.
4.4.2 Sample crushing: same as 3.4.2.
4.4.3 Preparation of test solution: Accurately weigh 2.0g of crushed and mixed sample (4.4.2) into a centrifuge tube, add 2mL water and 8mL ethyl acetate, oscillate and extract for 3min~5min, and centrifuge at 4000r/min for 1min. Take 2mL of supernatant into a glass beaker and blow dry the supernatant with an electric hair dryer. Add 0.4mL PBS buffer is used to dissolve the residue at the bottom of the beaker. This solution is the test solution. Note: According to the different limits of aflatoxin B in different varieties of grain in GB2761, different volumes of PBS buffer are selected to dissolve the residue at the bottom of the beaker. If the limit of aflatoxin B is 5μg/kg, 0.4mL PBS buffer is added to dissolve the residue at the bottom of the beaker; if the limit of aflatoxin B is 10μg/kg, 0.8mL PBS buffer is added to dissolve the residue at the bottom of the beaker: if the limit of aflatoxin B is 20μg/kg, 1.6mL PBS buffer is added to dissolve the residue at the bottom of the beaker. And so on.
4.5 Sample determination
Take 3~4 drops of the test solution (4.4.3) and slowly drip them into the sample addition hole of the test card (4.2.1), start timing, and read the results after 5 minutes. 2) The colloidal gold test card for aflatoxin B produced by Shanghai Kuailing Biotechnology Co., Ltd. and Beijing Huaan Mai Ke Biotechnology Co., Ltd. is currently the most widely used rapid test card. This information is provided only for the convenience of the users of this standard and does not represent the recognition of this product by this standard. Any test card that can meet the requirements of Appendix A of this standard can be used. 3
LS/T6108—2014
Note: The quality control color band must appear on the test card during each test. The quality control color band will appear within 2 minutes after adding the sample at the latest. 4.6
5 Nesting determination
4.6.1 Invalid
The quality control color strip does not show color, indicating that this test card is invalid and a new test card needs to be used for retesting. 4.6.2 Positive and negative samples
The quality control color strip shows color and the test color strip shows color, which is judged as negative, that is, the sample does not contain aflatoxin B or the aflatoxin B content is lower than the limit value set for this test.
When the quality control color strip shows color and the test color strip does not show color or the color is very blurred, it is judged as positive. That is, the aflatoxin B content in the sample is higher than the limit value set for this test.
When it is necessary to confirm the positive samples screened out by this method, the method specified in GB2761 should be used. 4
A.1 Technical requirements
A.1.1 Basic requirements
Appendix A
(Normative Appendix)
Technical requirements and verification methods for aflatoxin B in cereals, colloidal gold rapid test cardWorking environment requirements
Air temperature: 20℃~30℃.
2 Cross-reaction
LS/T6108—2014
No cross-reaction to deoxynivalenol toxin, zearalenone toxin, ochratoxin and other mycotoxinsA.1.2 Performance requirements
The appearance should meet the following requirements:
The surface of the test card should be smooth and flat, with uniform color, and the fiber membrane surface should not have defects such as exposed bottom, bubbles, pitting, wrinkles, brush marks, etc.;a)
The spot holes should be clean;
The font of words, symbols and other signs should be correct, clear and correct. c)
Accuracy
The error rate of the test results of the samples should not exceed 10%. For rice and brown rice samples with aflatoxin B content of 8.0rg/kg~12.0g/kg, and corn samples with aflatoxin B content of 16.0g/kg24.0μg/kg, the test results are allowed to have false positives, but not false negatives. Note: The test card has different test error ranges for different types of grains. For example, the test error range of the rice and brown rice sample test card is ±2/kg, and the test error range of the corn sample test card is ±4μg/kg. A.1.2.3 Stability
The error rate of the test cards of the same batch and different batches should not exceed 10%. A.2
Verification method
Positive sample value determination
The positive samples are determined according to the method specified in GB2761. A.2.2 Performance Test
A.2.2.1 Accuracy
When testing paddy, rice and brown rice samples, within the concentration range of 8.0ug/kg~12.0μg/kg aflatoxin B, select 10 samples with different concentrations for testing and calculate the error rate. 5
LS/T6108—2014
When testing corn samples, within the concentration range of 16.0ug/kg～24.0μg/kg aflatoxin B, select 10 samples with different concentrations for testing and calculate the error rate.
A.2.2.2 Stability
A.2.2.2.1
Batch-to-batch stability
Randomly select 3 batches of products, and use the same positive sample for at least 6 tests for each batch to calculate the error rate. 2 Intra-batch stability
A.2.2.2.2
Randomly select a batch of products and use the same positive sample to perform at least 6 tests to calculate the false positive rate. A.2.2.3 Product failure rate
The product failure rate during the shelf life should comply with the allowable value provided by the manufacturer. A.2.2.4 Agreed error range
The agreed error range should comply with the allowable value provided by the manufacturer.0μg/kg aflatoxin B, within the concentration range, select 10 samples with different concentrations for determination, and calculate the error rate. 5
LS/T6108—2014
When testing corn samples, within the concentration range of 16.0ug/kg～24.0μg/kg aflatoxin B, select 10 samples with different concentrations for determination, and calculate the error rate.
A.2.2.2 Stability
A.2.2.2.1
Batch stability
Randomly select 3 batches of products, and use the same positive sample for at least 6 determinations for each batch, and calculate the error rate. 2 Intra-batch stability
A.2.2.2.2
Randomly select 1 batch of products, and use the same positive sample for at least 6 determinations, and calculate the error rate. A.2.2.3 Product failure rate
The product failure rate during the shelf life shall comply with the allowable value provided by the manufacturer. A.2.2.4 Agreed error range
The agreed error range shall comply with the allowable value provided by the manufacturer.0μg/kg aflatoxin B, within the concentration range, select 10 samples with different concentrations for determination, and calculate the error rate. 5
LS/T6108—2014
When testing corn samples, within the concentration range of 16.0ug/kg～24.0μg/kg aflatoxin B, select 10 samples with different concentrations for determination, and calculate the error rate.
A.2.2.2 Stability
A.2.2.2.1
Batch stability
Randomly select 3 batches of products, and use the same positive sample for at least 6 determinations for each batch, and calculate the error rate. 2 Intra-batch stability
A.2.2.2.2
Randomly select 1 batch of products, and use the same positive sample for at least 6 determinations, and calculate the error rate. A.2.2.3 Product failure rate
The product failure rate during the shelf life shall comply with the allowable value provided by the manufacturer. A.2.2.4 Agreed error range
The agreed error range shall comply with the allowable value provided by the manufacturer.
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