GB 23748-2016 English PDFUS$399.00 · In stock
Delivery: <= 4 days. True-PDF full-copy in English will be manually translated and delivered via email. GB 23748-2016: Detection of irradiated foodstuffs -- DNA comet assay -- Screening method Status: Valid GB 23748: Historical versions
Basic dataStandard ID: GB 23748-2016 (GB23748-2016)Description (Translated English): Detection of irradiated foodstuffs -- DNA comet assay -- Screening method Sector / Industry: National Standard Classification of Chinese Standard: C53 Word Count Estimation: 20,283 Date of Issue: 2016-12-23 Date of Implementation: 2017-06-23 Older Standard (superseded by this standard): GB/T 23748-2009; NY/T 2214-2012; SN/T 2910.2-2011 Regulation (derived from): National Health and Family Planning Commission Notice No.17 of 2016 Issuing agency(ies): National Health and Family Planning Commission of the People's Republic of China, State Food and Drug Administration GB 23748-2016: Detection of irradiated foodstuffs -- DNA comet assay -- Screening method---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.Detection of irradiated foodstuffs - DNA comet assay - Screening method National Standards of People's Republic of China National Food Safety Standard Identification of irradiated food screening Issued on. 2016-12-23 2017-06-23 implementation National Health and Family Planning Commission People's Republic of China China Food and Drug Administration released ForewordThis standard replaces GB/T 23748-2009 "Identification of irradiated food were screened DNA comet assay method", NY/T 2214- 2012 "Identification of irradiated food OSL Law" and SN/T 2910.2-2011 "Identification of irradiated food outlet - Part 2. Single Cell Gel electrophoresis. " This standard compared with GB/T 23748-2009, the main changes are as follows. --- Standard name was changed to "national food safety standard screening method to identify irradiated foods"; --- Adds two screening methods. optically stimulated luminescence method and microbiological screening method; --- Increased DNA comet assay method of limiting instructions. National Food Safety Standard Identification of irradiated food screening1 ScopeThis standard specifies three rapid screening of food irradiation had accepted identification methods. optically stimulated luminescence method, DNA comet assay method and microorganisms Learning was screening. This standard OSL law applies to crustaceans, spices and condiments irradiation identification products; DNA comet assay method is suitable for Animal products, identification of irradiated grains, nuts, fruits and vegetables; microbiological screening method is suitable for frozen Poultry and Seafood, and other fresh food Irradiation identification. The first method OSL method2 Terms and definitions2.1 OSL (photostimulatedluminescence, PSL) After the rich silicate sample capture radiant energy by certain wavelengths of light excitation, in the form of light released from the phenomenon. 2.2 PSL intensity (PSLintensity) After the amount of light emitted by the sample is detected by optical excitation, the photon count rate indicates. 2.3 Screening PSL (screeningPSL) PSL initial strength of the sample measurement. 2.4 Calibration PSL (calibratedPSL) After screening PSL measurement, the same sample with a known dose of radiation after the PSL intensity measurement. 2.5 threshold (thresholds) In the screening mode, for determining whether or not a sample irradiated PSL strength, comprising a lower threshold (T1) and a high threshold (T2). 2.6 negative PSL (negativePSLresult) PSL measured intensity below the low threshold value. 2.7 Suspicious PSL (intermediatePSLresult) PSL measurement of strength between the low threshold and high threshold. 2.8 positive PSL (positivePSLresult) PSL intensity measured above the high threshold. 2.9 blank (darkcount) When no light stimulation, empty sample containers obtained photon count rate. 2.10 positive control (lightcount) The reference light source (such as a scintillator containing 14C or equivalent) placed photon count rate of the sample container obtained. Principle 3 Rich silicate material samples can be stored ray energy. Can excited by certain wavelengths of light irradiation again, the stored sample The amount can be released, resulting in the release of an optical signal light. Use light intensity of light release detector test light signal, large signal intensity comparison of different light Small, it is determined whether the samples irradiated.4 instruments and equipment4.1 OSL measurement system. including sample containers, excitation light source, and synchronous pulse stimulator photon counting system. 4.2 Measurement disc. diameter 5cm. 4.3 sources of ionizing radiation. ionizing radiation sources used and managed according to GB 17568 and GB/T 25306 requirements. Step 5 Analysis Note. Place the sample in the dark before measuring. Use disposable measuring disc is measured. 5.1 Preparation of spices and condiments samples Prepare duplicate samples, spread evenly in the bottom of the measuring plate were detected. If the two test results compared with the determination threshold value is inconsistent, then After re-detected sample aliquot 4, whichever is the highest value as a result of two detection determination, and so on. 5.2 Preparation of samples crustaceans The shell or shelled into the test sample dish, help detect the OSL signal when samples with animal intestines. As larger individuals, The samples can be cut; also desirable intestines (less than 6) in a measuring plate is measured. 5.3 Instrument settings Screening for irradiated food system set individual measurement parameters. Check the blank and positive controls. NOTE. should periodically or when a positive result after the measurement, the containers are empty sample testing, inspection equipment for contamination. 5.4 Determination of the sample 5.4.1 Screening Measurement Conduct sample testing and the results recorded. 5.4.2 Calibration Measurement Screening sample measured after capping save to prevent sample loss or contamination. These samples were then irradiated specific dose (spices and shellfish food Was irradiated 1kGy), room temperature (crustaceans and other perishable samples should be refrigerated after irradiation) from light 12h, calibration measurements.6 The results presentation6.1 threshold setting Spices and condiments threshold value is set. T1 = 700counts/min, T2 = 5000counts/min. Crustaceans threshold set as. T1 = 1000counts/min, T2 = 4000counts/min. 6.2 Analyzing Results According to the comparison with the threshold value, the measurement results of the sample can be divided into negative, suspicious, positive three cases were judged. 6.2.1 negative results 6.2.1.1 Screening PSL PSL screening result was negative, non-irradiated sample can be determined. 6.2.1.2 Calibration PSL When corrected PSL and PSL with negative screening, the sample can not be determined to be irradiated food. To further confirm whether the sample is then Received radiation should be used other confirmatory methods of identification of irradiated food. Calibration PSL negative, but positive screening PSL, indicating that the measuring system error, it should take a fresh sample of the original re-measured. 6.2.2 Suspicious Results 6.2.2.1 Screening PSL PSL as suspicious at screening results can not be directly determined whether the sample had received radiation. 6.2.2.2 Calibration PSL When corrected PSL and PSL screening results with the suspect, judging samples received irradiation. When corrected PSL as suspicious results PSL screening results are negative, indicating that the sample may be hypoallergenic non-irradiated foods. To enter a Step confirm whether the sample had received radiation, other confirmatory method should be used for identification of irradiated food. Calibration PSL as suspicious results PSL screening for high value positive results, indicating that the measurement is incorrect. Calibration PSL as suspicious results, positive results of screening PSL close to T2, indicating that the sample may have received higher doses of radiation correction Photos. It should be re-measured sample is determined. 6.2.3 positive results 6.2.3.1 Screening PSL PSL screening when a positive result, the sample can be determined received irradiation. 6.2.3.2 Calibration PSL When corrected PSL and PSL screening for a positive result at the same level judgment sample received irradiation. Calibration PSL when a positive result, and the result is far higher than the negative or suspicious screening PSL, indicates that the sample may not irradiated. Calibration PSL and PSL screening results were positive, but less than the screening correction PSL PSL (a difference of 1-2 orders of magnitude), indicating that the measurement Error, need to re-measure. The second method DNA comet assay method Principle 7 DNA molecules after irradiation fracture occurs. The cells were embedded in agar with lysis reagent dissolve the cell membrane, and then some Voltage electrophoresis. Damaged DNA fragments move quickly in the direction of the electric field to the anode, forming a tail-like distribution. After staining, DNA that is Cell loss occurs comet-shaped electrophoresis patterns, non-irradiated cells nearly circular pattern or a slight hangover. 8 kit Unless otherwise indicated, the methods used were of analytical grade reagents and water for the three water GB/T 6682 regulations. 8.1 dimethyl sulfoxide (DMSO, C2H6OS). 8.2 Sodium chloride (NaCl). 8.3 Potassium chloride (KCl). 8.4 Disodium hydrogen phosphate (Na2HPO4 · 12H2O). 8.5 potassium dihydrogen phosphate (KH2PO4). 8.6 disodium edetate (Na2EDTA, C10H14N2O8Na2 · 2H2O). 8.7 Sodium hydroxide (NaOH). 8.8 Tris (Tris, C4H11NO3). 8.9 boric acid (H3BO3). 8.10 sodium dodecyl sulfate (SDS, C12H25O4NaS). 8.11 Acridine Orange (C17H19N3 · HCl · ZnCl2). 8.12 ethidium bromide (C21H20BrN3). 8.13 trichloroacetic acid (TCA, C2HCl3O2). 8.14 zinc sulfate (ZnSO4). 8.15 glycerol (C3H8O3). 8.16 sodium carbonate (Na2CO3). 8.17 ammonium nitrate (NH4NO3). 8.18 silver nitrate (AgNO3). 8.19 tungstosilicic acid (H4 [Si (W3O10) 4] · xH2O). 8.20 Formaldehyde (CH2O). 8.21 Glacial acetic acid (CH3COOH). 8.22 sulfonated pyridine (C10H12O4N2S). 8.23 agarose. 8.24 low melting point agarose. Reagent preparation 9 Test used in the reagent preparation see Appendix A. 10 instruments and equipment 10.1 horizontal electrophoresis tank. 10.2 Electronic balance. a sense of the amount of 0.1g. 10.3 Electric heating magnetic stirrer. 10.4 microwave. 10.5 porous membrane. pore size 100μm, 200μm, 500μm. 10.6 microscope slides. 76mm × 26mm, with frosted edges. 10.7 fluorescence microscope. 100 to 400-fold; blue excitation wavelength of 460nm ~ 485nm, for acridine orange staining; green excitation wave Length 515nm ~ 560nm, for sulfonated pyridine or ethidium bromide staining. 11 analysis steps 11.1 Sample Preparation 11.1.1 animal tissues 11.1.1.1 marrow sample preparation Cut bone marrow weigh about 50mg a test tube, add 3mL ice-cold PBS buffer. Stirring with a glass rod, the fine Hanging from the cell, the cell suspension was filtered with a microporous membrane 100μm, and the filtrate was collected and the filtrate was placed on ice boxes, alternate (cell suspension can be placed in ice Placed, no more than 10min; after adding 5% to 10% DMSO as antifreeze, the cells can be placed at -20 ℃ 26h). 11.1.1.2 muscle tissue sample preparation Muscle tissue with a scalpel to cut into thin slices (not visible fat). Weigh 1g muscle placed in a small beaker, add ice-cold 5mL PBS buffer. The beaker was placed in an ice box, a glass rod stirring 5min, followed by 500μm and 200μm microporous membrane filtration, The filtrate was collected and placed 5min, on standby in the ice box. 11.1.2 Plant Tissue 11.1.2.1 raw grain, nuts and other food seasonings Sample Weigh 0.25g, ground with a mortar, placed in a small beaker, add 3mL ice-cold PBS buffer. The beaker was placed in an ice Box with a glass rod stirring for 5min, followed by 500μm and 200μm microporous membrane filtration, the filtrate was collected and placed in an ice box 15min ~ 60min, standby. 11.1.2.2 strawberries and other berries Weigh 0.25g strawberry berry, ground with a mortar, placed in a small beaker, add 3mL ice-cold PBS buffer. The beaker is set Ice box with a glass rod stirring for 5min, followed by 500μm and 200μm microporous membrane filtration, the filtrate was collected and placed in an ice box 15min ~ 60min, standby. 11.1.2.3 vegetables (not including edible fungi) The edible part of vegetables sliced with a scalpel, take 4g, was added 5mL ice-cold PBS buffer, ground with a mortar and placed Small beaker, add 3mL ice-cold PBS. The small beaker placed in an ice box, a glass rod stirring 5min, followed by 500μm and 200μm microporous membrane filtration, the filtrate was collected and placed 15min ~ 60min, standby on an ice box. 11.2 precoated slides Prior to coating, the slides should be soaked with methanol to remove surface oil. After air drying, a drop (approximately 50μL) agarose solution in the carrier coating On the slide, immediately take another cover slides on agarose, agarose solution so spread out, remove the upper layer slides, air-dried 30min, and set aside. 11.3 embedding gel Take 100μL cell suspension was mixed with 1mL embedded gel solution, and then take the mixture with a micropipette 100μL pipette tip light Light coated on a pre-coated glass slides, coverslipped immediately spread the gel evenly, careful not to create a bubble. The slides were placed on ice box 5min, then peel off the cover slip from the agarose using a dissecting tip. 11.4 cells were lysed The slides were immersed in 11.3 fully prepared lysis buffer (animal cell ≥5min, plant cells ≥15min), the cells were dissolved Solution, do not touch agarose dissolution process. 11.5 rewet The slides were immersed in the running buffer, time 5min. 11.6 electrophoresis The slides were placed side by side horizontal electrophoresis tank, scrub edge toward the cathode. Electrophoresis tank electrophoresis buffer was added not too slides 2mm ~ 4mm. Electrophoresis at room temperature for 20min, voltage 2V/cm. After turning off the power, the slides into the water 5min, air-dried at room temperature for 1h. 11.7 dyeing 11.7.1 acridine orange, sulfonated pyridine or ethidium bromide staining The slides were immersed in staining solution (acridine orange staining solution, sulfonated pyridine or ethidium bromide staining solution staining solution) 3min ~ 5min, and then immersed Water cleaning 0.5min ~ 1min, wipe excess water slides under fluorescence microscope. 11.7.2 silver staining The slides were placed in a mixture of A10min, washing 1min, 40 ℃ ~ 50 ℃ constant temperature drying oven to dry at room temperature or 1h. then The slides were immersed in a mixture of D10min ~ 20min, repeat the steps 1 to 2 times, dyeing time 5min ~ 10min, until slides Rendering brown. Washing 1min, with a stop solution soak 5min E staining reaction is stopped, and then washed with water 1min. Finally, the slides were placed at room temperature, Dry naturally. Stained slides will not fade, can be stored for a long observation. Note. The fluorescent dye easily quenched rapidly to staining; staining solution toxic, after the end of the test, to deal with the waste disposal purification treatment again. Observation 11.8 Identification 11.8.1 fluorescence observation Acridine orange stained slides using fluorescence microscopy (460nm ~ 485nm, blue light excitation) observed, staining DNA emits green Fluorescence. Sulfonated pyridine or stained with ethidium bromide fluorescence microscope slides (515nm ~ 560nm, green excitation) with 590nm filter Rays observed red fluorescence staining DNA. 11.8.2 White Observation Silver nitrate stained slides can be observed using an ordinary microscope. 12 analysis results presentation 12.1 Expression of results Through the microscope, the irradiated sample almost no intact cells, all comet image (see Figure B.1 and B.2); undamaged Cells do not have light or tailing tailing (see Figure B.3 and B.4). 12.2 determination result When the image appears comet, results are reported as positive, the sample may be irradiated. When the comet image does not appear, the result is reported as negative, non-irradiated samples. 13 limiting illustration In fact the comet samples to produce different shapes will be significantly different, but other processes may also affect DNA form, which gives the convergence According to the determination of food difficult. Therefore, DNA comet assay as a screening method can only approach irradiated foods, and ultimately determine whether to accept food Irradiation, irradiated foods need to meet the other confirmatory methods for testing. The third method microbiology screening 14 Terms and Definitions Endotoxin (endotoxin) General toxic substances in the presence of gram-negative bacteria cell is endemic on the bacterial cell wall, and its toxic components primarily for the class Lipid A. Only after lysis of bacteria, endotoxins will be released, the host can cause fever and endotoxin shock and other symptoms. Principle 15 Under suitable conditions, bacterial endotoxin activates the pro-clotting enzyme in LAL, LAL make produce agglutination reaction to form a gel. Endotoxin Gram-negative bacteria was positively correlated with the content, so the content can be determined according to the content of Gram-negative bacteria endotoxin. Food irradiation after a dose of food in Gram-negative bacteria are killed substantially, but not because of residual endotoxin exposure Treatment disappears. Therefore, the concentration of endotoxin can be measured to calculate the total number of irradiated foods dead and alive Gram-negative bacteria, while Surviving on food Gram-negative bacteria were cultured counted by the difference between the two, it can determine whether irradiated food. If the two numbers The obvious difference value, indicating that the sample may have received radiation. 16 Reagents and materials 16.1 Reagents 16.1.1 LAL. Sensitivity 0.5EU/mL. 16.1.2 non-pyrogenic saline injection. 16.2 Materials 16.2.1 peptone solution (see C.1). 16.2.2 nutrient agar (see C.2). 16.2.3 milk agar (see C.3.1). 16.2.4 crystal violet solution (see C.3.2). 16.2.5 penicillin solution (see C.3.3). 16.2.6 Gram-negative bacteria selective medium (see C.3.4). 16.3 Standards Endotoxin standard sample (CAS No. 1235503). 50EU/tube, purity ≥99%. 16.4 Standard Solution Endotoxin standard solution. take endotoxin standard sample, no heat is added 1mL saline dissolution of 50EU/mL of the standard solution. 17 instruments and equipment 17.1 thermostat water bath. 17.2 Autoclave. 17.3 Electronic balance. a sense of the amount of 0.1g. 17.4 oven thermostat. 17.5 Vortex. 17.6 Heated Incubators. 17.7 homogenizer. 8000r/min ~ 10000r/min. 18 analysis steps Note. Samples should be stored at 4 ℃ within 24h test. If you can not verify immediately, it should be placed at -20 ℃, the longest storage time is not more than 30d. 18.1 sample preparation Under sterile conditions, in different parts of the sample of the sample, for a total 10g. The sample was placed in a sterile plastic container using a disposable syringe added 90mL nonpyrogenic saline. 8000r/min homogeneous 1min ~ 2min, to a concentration of the sample solution 10-1, 4 ℃ stored for use, Maximum storage time is not more than 24h. 18.2 Gram-negative bacterial culture counts 18.2.1 media preparation Preparation of peptone solution, nutrient agar, Gram-negative bacteria selective medium (see Appendix C), pour the melted front plate Training based on 45 ℃ ± 1 ℃ water bath insulation. 18.2.2 dilution and culture 18.2.2.1 The sample contamination of the sample solution (10-1) further diluted with peptone solution for sample dilution in accordance with 9mL 1mL volume ratio than 10-fold dilution, the sample diluent to give a concentration of 10-2 to 10-5. 18.2.2.2 The coated sample diluent 0.1mL different dilutions in nutrient agar plates at room temperature, allowed to stand for 90min up front. Each dilution do two plates. 18.2.2.3 Each plate covering 10mL Gram-negative bacteria selective medium. Horizontally, so that solidification of the agar. Invert t......Tips & Frequently Asked Questions:Question 1: How long will the true-PDF of GB 23748-2016_English be delivered?Answer: Upon your order, we will start to translate GB 23748-2016_English as soon as possible, and keep you informed of the progress. The lead time is typically 2 ~ 4 working days. The lengthier the document the longer the lead time.Question 2: Can I share the purchased PDF of GB 23748-2016_English with my colleagues?Answer: Yes. The purchased PDF of GB 23748-2016_English will be deemed to be sold to your employer/organization who actually pays for it, including your colleagues and your employer's intranet.Question 3: Does the price include tax/VAT?Answer: Yes. 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