GB/T 23748-2009Detection of irradiated foodstuffs -- DNA comet assay -- Screening method (English PDF)

2.1 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.

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 presentation

6.1 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.

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 °C 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, 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 °C ~ 50 °C 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; 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.

12.1 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 (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.

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.

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 °C 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 °C ± 1 °C 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 the plate on 21 °C ± 1 °C incubator for 24h ± 1h.

18.2.2.4 selected colonies of two consecutive dilutions plate count of 15 to 300 (see Table 1 Example D.1). Per gram of sample Gram Negative bacteria according to equation (1).

N = sum

V × n1 0.1 × n2 () [] × d (1) where.

N --- gram negative bacteria per gram of the sample, in units of colony forming units per gram (CFU/g);

sumc --- long or 15 to 300 colonies of two successive dilutions and plate the colonies;

The V --- volume of broth per plate inoculated in milliliters (mL of);

Count the number of flat n1 --- first dilution;

Count the number n2 --- flat second dilution;

d --- plate count valid first dilution.

If only a dilution plate length 15 to 300 colonies, then n2 counted as 0 (see Table D.1 Example 2).

If the plate count all dilutions are less than 15, or the highest number of colonies flat estimates as N values (see

Table D.1 Example 3).

If all dilutions of colony no longer bacteria, results were expressed as gram-negative bacteria is not detected, N value as 0 (see Table D.1

Example 4).

Within 18.3 endotoxin detection

18.3.1 LAL detection

18.3.1.1 LAL dissolved and dispensing

According to the labeled amount of reagent was added pyrogen saline solution, and mix with each 0.1mL bottle packed in amperes.

18.3.1.2 Sample dilution

Use 96-diluted sample. Each well was added a 650µL sample was diluted in pyrogen-free saline. Take 300µL sample solution A diluted sample is added to the first wells, mixed uniformly, a sample diluent to obtain 10-0.5. The 300µL10-0.5 sample diluent was added to the first Samples were diluted two wells, sample diluent to give 10-1. Then 300µL10-1 sample diluent was added to the third sample dilution holes, Get 10-1.5 sample dilution. Continue diluted the sample until a final dilution of the toxin test is negative (see Figure D.3). finally the dilution depends on sample availability.

Diluted toxin standard solution within 18.3.1.3 Formulated within good endotoxin standard solution in accordance with 18.3.2.2 sample dilution different multiples.

18.3.1.4 LAL detection

The reaction carried out in 96-well plates. Within endotoxin standard solution was used as a positive control, non-pyrogenic saline as a negative control. Each Wells diluted sample was added 0.1mL and 0.1mL LAL, and mix. Cover with a lid 96, placed 37 °C ± 0.5 °C water bath the reaction 1h.

18.3.2 Results count

When the reagent occurs completely aggregated as "" part when aggregated as "/ -" when no agglutination as "-." Endotoxin within 18.3.3 LAL detection sensitivity is determined based on the detection of the situation within the different dilutions of endotoxin standard solution. In the last solidified Samples used to calculate the titer of the diluted solution, if completely solidified, is used to calculate the actual titers (see Table D.2 in Example 1), if the Department of Solidification points "/ -" Later, the places of the index minus a multiple of 0.25 as a value used to calculate the titer (see Table D.2 in Example 2). Per gram of sample Endotoxin content according to formula (2).

LAL =

c × 10t × 10

10T

Where.

Within LAL --- endotoxin content per gram of sample units of endotoxin units per gram (EU/g);

C --- inside the standard endotoxin units of endotoxin units per milliliter (EU/mL);

--- t titer of the sample;

T --- standard dilution.

19.1 Analysis

19.1.1 Calculation lgLAL, lgN and lgLAL-lgN value.

19.1.2 lgLAL-lgN > 0, indicates that the sample tested contains a high content of endotoxin, but the number of Gram-negative bacteria than the cultured There are not detected or less can be cultured Gram-negative bacteria.

19.1.3 lgLAL-lgN value <=0, indicates that the sample tested contains a high content of endotoxin, and the number of gram-negative bacteria cultured More.

19.1.4 lgLAL 0, it is determined that the sample may after irradiation.

19.2.2 lgLAL-lgN value <=0, determined that the sample has not been irradiated.

19.2.3 LgLAL Appendix A

The method of reagent preparation A.1 hydrochloric acid (1mol/L) Measure 90mL of hydrochloric acid, diluted with an appropriate amount of water to 1000mL.

A.2 PBS buffer (pH 7.4) Weigh 8.0g sodium chloride, 0.2g KCl, 2.94g disodium hydrogen phosphate dodecahydrate, 0.24g potassium dihydrogen phosphate dissolved in 900mL water Mix well, adjust the pH with hydrochloric acid (A.1) = 7.4, then dilute to 1000mL, after autoclaving spare.

A.3 coating agarose solution (0.5%) 10mL of distilled water was added 50mg of agarose, boil or microwave heating, 45 °C water bath until needed.

A.4 embedded gel solution (0.8%) 10mLPBS (A.2) was added 80mg low melting point agarose, boil or microwave heating, 45 °C water bath until needed.

A.5 sodium hydroxide solution (40%) Weigh 40g of sodium hydroxide dissolved in 60mL water.

A.6 EDTA stock solution (0.5mL/L) Weigh 93.05g Disodium edetate was dissolved in 300mL of water, mixed with sodium hydr...