GB 5009.261-2016National food safety standard - Determination of neurotoxic shellfish poisoning in shellfish (English PDF)

3.1 Reagents

3.1.1 Hydrochloric acid (HCl).

3.1.2 Anhydrous ether (C4H10O).

3.1.3 Tween-60 (C64H126O26).

3.1.4 Sodium chloride (NaCl).

3.1.5 Sodium hypochlorite (NaClO).

3.2 Reagent preparation

3.2.1 Sodium chloride solution (0.85%). weigh 0.85 g of NaCl, add water to

dissolve and set volume to 100 mL.

3.2.2 1%Tween-60. weigh 1.0 g of Tween-60, use 0.85% sodium chloride

solution to dissolve and set volume to 100 mL.

3.2.3 Sodium hypochlorite solution (5%). weigh 50 g of sodium hypochlorite,

use water to dissolve and dilute to 1000 mL.

3.3 Standard product

Short naked toxin analog (brevetoxin-2), purity >= 90%.

3.4 Materials

Mouse. healthy ICR strain male mouse weighing 19g ~ 21g.

4.1 Rotary evaporator.

4.2 Homogenizer.

4.3 Balance. resolution of 0.1 g.

4.4 Separating funnel.

4.5 Round-bottomed flask.

4.6 Brinell funnel.

4.7 Disposable syringe. 1mL.

4.8 Centrifuge. rotating speed >= 6000 r/min.

4.9 Metal screen. aperture of about 2mm.

5.1 Specimen preparation

5.1.1 Sample collection

At least 10 shellfish shall be taken for each analysis sample to make the

shellfish more than 200g.

The frozen sample is placed in an insulation box sent for inspection. Or ensure

it is at a low temperature state (0°C ~ 10°C) sent for inspection. If it is a shell

sample, it shall be opened to remove moisture and then sent for inspection.

5.1.2 Specimen preparation

5.1.2.1 Fresh shell sample

Use clean water to wash the shell surface thoroughly. Cut off the muscle. Open

shell. Use water to rinse the interior so as to remove sediment and other foreign

objects. Separate the occipital muscle from the tissue attached to the gluing.

Take out the shellfish. Never cut the flesh. Do not heat or use anesthetic before

opening the shell. Collect 200g of shellfish and drain it on a metal sieve with a

pore size of about 2mm for 5min. Pick up broken shells and other debris.

Homogenize the shellfish, for use.

5.1.2.2 Frozen sample

At room temperature, make the frozen sample semi-frozen. For shell frozen

sample, according to the method in 5.1.2.1, clean, open shell, rinse and take

the meat. Remove the borneol attached to the outside of the shellfish, wipe off

the water, and then relax at room temperature. Collect 200g of shellfish and

drain it on a metal sieve with a pore size of about 2mm for 5min. Homogenize

the shellfish, for use.

5.1.2.3 Canned shellfish

Drain the contents of the can. Pour into the homogenizer for full homogenization,

for use.

5.1.2.4 Dried shellfish product

Weigh 100g of dried product into sufficient water, soaking 24h ~ 48h

(refrigerated at 4°C). Drain, homogenize, for use.

5.1.2.5 Salted product

Use clean water to rinse. Use running water to desalt. Drain, homogenize, for

use.

5.2 Specimen extraction

Take 100g of specimen into a 500mL beaker. Add 5g of sodium chloride and

1mL of concentrated hydrochloric acid. Mix them well. While stirring, heat the

mixture to it is boiling. Boil it on a slow fire for 5min. Cool to room temperature.

Transfer the mixture to a 500mL centrifuge tube. Use 50mL of ether to rinse the

beaker. Move the rinse together into the centrifuge tube. Add 100mL of ether

into the centrifuge tube. Cap a plug. Shake thoroughly. Centrifuge at 6000 r/min

for 15min.

After centrifugation, carefully pour off the ether layer (upper layer) solution into

a 1000mL separatory funnel. Use ether to repeat the extraction of the

precipitate in the centrifuge tube three times. The total amount of extraction for

three times is 250 mL of ether. Transfer the ether layer liquid to the separatory

funnel. Gently oscillate (cannot produce emulsion). Remove the water layer

(lower layer) and shellfish fragments after static layering and stratification.

Transfer the ether layer to a 500mL round bottom flask. Conduct concentration

under reduced pressure (rotary evaporator, 35°C ± 1°C) to remove ether.

Use 1%Tween-60 saline to dilute the concentrate to 10 mL in a graduated tube.

Make a full shake. Prepare it to a uniform NSP-1%Tween-60 saline suspension.

At this time, the amount of 1 mL of liquid corresponds to the weight of 10 g of

shelled shellfish measured in advance. Take this suspension as test stock

solution for animal experiment.

5.3 Mouse test

Choose 6 healthy ICR strain male mice with a body weight of 19g ~ 21g. Divide

them into 2 groups randomly. testing sample group and blank control group

(1%Tween-60 saline), 3 mice/group.

Separately take 1 mL of testing solution or 1%Tween-60 saline for

intraperitoneal injection. If the extract overflows during the injection, the mouse

must be discarded and another mouse shall be re-injected. Record the time of

completion of injection and the time of death when the mouse stops breathing,

and observe 930min continuously. If the median death time of the mice is within

2h, the test solution shall be diluted with 1%Tween-60 saline to re-inject until

the death time of the mice is within 2h ~ 6h.

6.1 Determination of the corrected mouse unit (CMU) of testing sample

According to the mouse death time of the testing sample (see Table A.1), the

corresponding mouse unit number MU is found. Find the corresponding body

weight correction coefficient based on the weight of the mouse (see Table A.2).

The mouse unit of the same mouse is multiplied by the body weight correction

coefficient to obtain the CMU of this mouse. Select the median CMU of 3 mice

in the test group, which is the median CMU of the sample group. Use this value

to conduct the calculation of 6.2.

6.2 Calculation of toxin virulence and expression of results

6.2.1 NSP virulence calculation

The NSP virulence in the sample is calculated according to formula (1).

where,

X - NSP virulence in the sample, in mouse unit per gram (MU/g);

CMU - Median correction mouse unit of the testing group of the mice of testing

sample, in mouse unit per milliliter (MU/mL);

DF - Dilution multiple.

NOTE. 10 - the unit is milliliter; 100 - the unit is gram.

6.2.2 Expression of results

In the case where the blank control mice are normal, the following judgments

and expressions are made.

If the death time of the mouse is equal to 360min, the NSP virulence of the

testing sample is equivalent to 0.2 MU/g.

If the median death time of the experimental group is less than 120min, the

sample extract shall be diluted. Select another 3 mice for the test till the median

death time is 120min ~ 360min. Calculate the mouse unit virulence of the

sample based on the final dilution test results. Report the NSP virulence of this

sample as. XXX MU/g.

If the median death time of the experimental group is greater than 360min,

directly calculate and determine the virulence of the sample mouse. Report the

NSP virulence of this sample as. XXX MU/g.

If all the mice in the experimental group do not die within 930min of observation,

the NSP virulence of the sample shall be also reported as less than 0.1 MU/g.

Annex A

Conversion between mouse death time and mouse unit

Refer to Table A.1 for the conversion between mouse death time and mouse

unit.

Table A.1 -- Conversion between mouse death time and mouse unit

Table A.2 -- Correction coefficient for mouse weight

Death time / min Mouse unit / MU

Mouse weight / g Weight correction coefficient

Table A.2 (continued)

Mouse weight / g Weight correction coefficient

......