NY/T 2550-2014 Determination of aflatoxin B1 in feeds-Colloidal gold method NY/T2550-2014 |tt||Standard compressed package decompression password: www.bzxz.net

ICS65.120
Agricultural Industry Standard of the People's Republic of China
NY/T2550—2014
Determination of aflatoxin B, in feed--Colloid gold method2014-03-24 Issued
2014-06-01 Implementation
Ministry of Agriculture of the People's Republic of China
This standard was drafted in accordance with the rules of GB/T1.1—2009. This standard was proposed and managed by the Ministry of Agriculture of the People's Republic of China. NY/T2550--2014
The drafting units of this standard are: Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Oil Crops and Products Quality Supervision and Inspection and Testing Center of the Ministry of Agriculture. The main drafters of this standard are: Li Peiwu, Jiang Jun, Zhang Qi, Xiaoxia, Zhang Wen, Zhang Zhaowei, Wang Du, Tang Xiaoqian. E
1 Scope
Determination of aflatoxin B in feed Colloidal gold method This standard specifies the method for determining aflatoxin B in feed by colloidal gold method. This standard is applicable to the determination of aflatoxin B in feed and feed raw materials. The detection limit of aflatoxin B in this standard is 1.0μg/kg. 2 Normative references
NY/1 2550—2014
The following documents are indispensable for the application of this document. For any dated referenced document, only the dated version applies to this document. For any undated referenced document, the latest version (including all amendments) applies to this document. GB/T6682 Specifications and test methods for water used in analytical laboratories 3 Principle
During the chromatography process, aflatoxin B in the sample binds to the specific antibody labeled with colloidal gold. The immune reaction between the antibody and the aflatoxin B-3SA conjugate on the cellulose ether membrane detection line is inhibited, making the detection line lighter in color, and the determination is made by detecting the color change. 4 Reagents
Unless otherwise specified, only analytically pure reagents are used. 4.1 Water, grade -1 according to the requirements of GR/T6682. 4.2 Sucrose (C2H22O).
4.3 Bovine serum albumin (TSA) with a purity greater than 98% 4.4 Tween-20 (C%H:O2;).
4.5 Methanol (CH.OH).
4.6 70% methanol solution: 70.0 ml of colloid. Tween (4.5). Add 30.0 ml of water (4.1) and mix. 4.7 Sample dilution: Take 1.0g of sucrose (4.2), 0.5g of bovine serum albumin (4.3) and 2.5g of pyrrolidone-20 (4.4) and dissolve them in 100.0ml of water (4.1). 4.8 1000ng/ml, standard solution of aflatoxin B. 4.9 100ng/ml. Standard stock solution of aflatoxin B: accurately pipette 1.0ml of aflatoxin B: standard solution (4.8). 10ml of ethanol. Volumetric flask. Store at 4℃ for 3 months. Warning: Aflatoxin is highly toxic - test personnel should pay attention to self-protection. During operation, avoid inhalation and contact with aflatoxin standard solution. The standard solution should be prepared in a fume hood. Wear glasses, work clothes and medical latex gloves when working. All containers that come into contact with aflatoxin must be immersed in 10% sodium hypochlorite solution for more than 12 hours. At the same time, in order to reduce the chance of contact with aflatoxin, this standard encourages the purchase and use of certified standard stock solutions of aflatoxin. 5 Instruments and equipment
5.1 Spectral imaging detector or colloidal gold immunochromatography detector: image resolution better than 2048×1532dpi. 5.2 Analytical balance: sensitivity 0.1g,
5.3 Sample sieve: 20 months.
5.4 Homogenizer, speed greater than or equal to 20000r/minNY/T2550—2014
5.5 Vortex mixer.
5.6 Constant temperature device: (37.0=2.0)℃:
5.7 Micropipette: 1 μL_-~10 μl, 10μl-~100μL, 100μl.1 000 μl-. 5.8 Aflatoxin B colloidal gold immunochromatography device (Figure 1): fixed aflatoxin B:-BSA conjugate, detection sensitivity is not less than 0.3μu/kg. Carrier used for the reaction of aflatoxin and aflatoxin B,-BSA conjugate with colloidal gold labeled antibody in samples. Description:
(a) Regular diagram
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Water absorption;
Inspection diagram:
Gold potential:
b Measurement diagram
A sample pad;
Control line;
Figure 1 Aflatoxin B colloidal gold immunochromatography device 5.8.1 The aflatoxin B-BSA coupling ratio is 1:5)~(1:20) (BSAAFB). 5.8.2 Sample pad: glass fiber, polyester fiber or paper sheet 5.8.3 Nitrocellulose mold: 411 capillary time not less than 135s 5.8.4 Gold label pad: attached with 5ul colloidal gold labeled xanthotoxin B antibody. 5.8.5 Absorbent paper.
5.8.6 Bottom plate,
5.8.7 Connecting tape.
5.9 Medium-speed qualitative filter paper.
Purification column: 3 mL silica gel SPE tube.
6 Analysis steps
6.1 Sample preparation
Crush the sample until the edible part passes through the sample sieve (5.3) and mix thoroughly. 2
6.2 Pretreatment and preparation of blank matrix solution
6.2.1 Pretreatment
NY/T 2550--2014
Accurately weigh 25.0 g of sample and place it in a beaker. Accurately add 100 ml of ethanol solution (4.6). Use a homogenizer (5.4) to extract at 20000 I/min for 2 min. Let it stand for 1.5 min. Filter through medium-speed qualitative filter paper (5.9) and collect the filtrate. Take 2.5 ml of the filtrate and pass it through a purification column (5.10). Collect the purified solution. Use diluent (4.7) to dilute the purified solution to the detection range of aflatoxin B1 colloidal gold immunochromatography device (5.8), mix well with a vortex mixer (5.5) and set aside. 6.2.2 Preparation of blank matrix solution: Take a negative sample. Prepare a blank matrix solution according to 6.2.1 and set aside. 6.3 100 μl. Add the diluted purified solution (6.2.7) into the aflatoxin B colloidal gold immunochromatography device (5.8). React in a constant temperature device (5.6) for [0 min,
6.4 Detect on the machine (5.1).
6.5 Standard curve
Accurately pipette 0.000ml, 0.001ml, 0.005ml, 0.010ml, 0.020ml, 0.050ml, 0.100ml, 0.150ml of the standard stock solution of aflatoxin B into a 10ml volumetric flask. Make up to volume with blank matrix solution (6.1.2), which is equivalent to 0.00 ng/mL, 0.01 ng/ml, 0.05 ng/ml, 0.10ng/ml, 0.20 mg/ml, 0.50 ng/mL, 1.00ug/ml, 1.50 ng/ml-concentration standard working solution. Perform tests from low to high concentrations (6.3, 6.4), and establish a standard curve of aflatoxin B based on the ratio of the signal value of the test line T to the signal value of the quality control line (I/C) and the logarithm of the concentration of the standard working solution (logc). 7 Result calculation
7.1 Confirmation of the effectiveness of the colloidal gold immunochromatographic device A red stripe appears on the quality control line of the colloidal gold immunochromatographic device for aflatoxin B, which is considered to be effective. The results can be measured by visual inspection or spectral imaging detector or colloidal gold immunochromatographic detector (5.1); if the quality control line of the colloidal gold immunochromatographic device does not show a red stripe, is thick or seriously uneven, the colloidal gold immunochromatographic device is considered to be invalid and needs to be retested. 7. 2 Visual inspection method
If a red stripe appears on the detection line of the colloidal gold immunoassay device for aflatoxin B, it means that the content of aflatoxin 13 in the sample is less than its limit value, and it is judged as negative; if a red stripe does not appear on the detection line of the colloidal gold immunoassay device for aflatoxin B, it means that the content of aflatoxin B in the sample is greater than its limit value, and it is judged as positive. 7.3 Calculation and expression of results of instrumental method
The content of aflatoxin B in the sample is expressed as mass fraction X, and the value is expressed in micrograms per gram (ug/kg), calculated according to formula (1). X=VXn
Where:
p——The value of the content of aflatoxin B in the test solution obtained from the standard curve, in nanograms per milliliter (ng/ml.); V is the volume of the sample solution, in milliliters (mL); n is the dilution factor of the sample;
n is the mass of the sample, in grams (g). The calculation result is retained to one decimal place. 8 Precision
8.1 Repeatability
The instrumental method was used for determination. Under the repeatability conditions, when the aflatoxin B content was not more than 1.0.u/kg, the relative difference of two independent determination results should not exceed 20%; when the aflatoxin B content was greater than 10.0/kg, the relative sum difference of two independent determination results should not exceed 15%.
NY/T 2550-2014
Reproducibility
The instrumental method was used for determination. Under the reproducibility conditions, when the aflatoxin B content was not more than 10.0ug/kg, the relative difference of two independent determination results should not exceed 30%; when the aflatoxin B content was greater than 10.0ug/kg, the relative difference of two independent determination results should not exceed 20%.
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