SN/T 1162-2020Technical specifications for quarantine of infectious pancreatic necrosis (English PDF)

7.1 Sampling

The sampling quantity shall be implemented in accordance with GB/T 18088.For fish with clinical symptoms, take the whole fish fry less than or equal to 4 cm in length, if the yolk sac should be removed for fish with yolk sac; fish fry with a body length of 4 cm to 6 cm should be taken from internal organs (including kidneys); fish with a body length of more than 6 cm should be taken from liver, Kidney and spleen. Liver, kidney and spleen are taken from fish without clinical symptoms; ovarian fluid is also required for mature female fish. The egg shell is taken from fish eggs.

7.2 Sample processing

Should be kept below 10 °C for processing. First homogenize the sample with a tissue grinder, and then suspend it in the fine particles at a final dilution of 1.10.

Cell culture medium (see B.2 in Appendix B). If the sample has not been treated with antibiotics before homogenization, it must be added to the cell culture medium 1 000 IU/mL penicillin and 1 000 µg/mL streptomycin. Incubate at 15 °C for 2 h ~ 4 h or at 4 °C for 6 h ~ 24 h. 7 000 r/min Centrifuge for 15 min and collect the supernatant. The ovarian fluid does not need to be homogenized, it should be diluted more than 2 times. Centrifuge samples of ovarian fluid in the same way and later the supernatant was used directly in the step.

7.3 Virus isolation

Dilute the 1.10 tissue homogenate supernatant twice with cell culture medium by 10 times, and then dilute 1.10, 1.100, 1.1 000 3 the diluted supernatant was inoculated into CHSE-214, RTG-2 or CHSE-214, RTG-2 or in the BF-2 cell monolayer, each well (2 cm2) of the cell monolayer is inoculated with a maximum of 100 µL of diluent. After adsorption at 15 °C ~20 °C for 0.5 h ~1 h, Add cell culture medium. Incubate at 18 °C ± 2 °C.

After the positive control group and the test sample were inoculated with cells, they were inspected with an inverted microscope every day for 7 days. The blank control cells should be normal.

If, except for the positive control cells, there is no cytopathic (CPE) in the cell culture of the tested homogenate supernatant diluent, then culture 7 After d, it is necessary to subculture with sensitive cells. During subculture, freeze-thaw and collect the cell monolayer culture inoculated with the supernatant diluent of the tissue homogenate.

The nutrient was centrifuged at 7 000 r/min 4 °C for 15 min, and the supernatant was collected. Inoculate the supernatant into a fresh cell monolayer and culture for 7 days, every day Microscopic examination. If there is no CPE after blind transmission, the result is judged as negative.

If CPE appears in the cell culture inoculated with the supernatant diluent of the tested homogenate or the cell culture after blind transmission, it should be applied immediately SYBR Green real-time fluorescent RT-PCR, conventional RT-PCR or ELISA methods for IPNV identification. CPE appears as a local area the domain cells begin to lyse, the surrounding cells shrink and become round, gradually develop, and finally all the cells fall off.

If there is no CPE in the positive control, the test is invalid. You must switch to sensitive cells and a new batch of tissue samples according to the above method.

Method for virological examination.

7.4 SYBR Green real-time fluorescent RT-PCR detection of IPNV

7.4.1 Setting up a comparison

Blank control, negative control and positive control should be set for each reaction. For blank control, DEPC water can be used instead of sample; negative control it is a tissue or cell culture that does not contain IPNV; a positive control is a positive tissue or cell culture that contains IPNV or contains a fragment of interest RNA.

7.4.2 RNA extraction

Take.200 µL of the tissue supernatant of the sample to be tested or the cell culture of the sample to be tested, add 1 mL of Trizol reagent, and use a pipette Fully pipette 10 to 20 times, and place at room temperature for 5 minutes; add.200 µL of chloroform, vortex for 30 s to mix, and place at room temperature 15 min; centrifuge at 12 000 r/min for 10 min; take the upper water phase to a new centrifuge tube, add an equal volume of isopropanol, and turn upside down.

Mix it again and place it at -20 °C for 20 min; centrifuge at 12,000 r/min for 10 min; discard the supernatant, and wash the pellet with 1 mL of 75% ethanol;

Centrifuge at 8 000 r/min for 10 min, discard the supernatant, and dry the pellet at room temperature for 5 min; add 20 µL of DEPC water to dissolve the RNA pellet. 4 °C ice Keep in the box for later use. The RNA solution should avoid repeated freezing and thawing and be used for testing as soon as possible.

RNA extraction can also use equivalent commercial RNA extraction kits instead of the above methods.

7.4.3 Reaction system

In a 0.2 mL PCR reaction tube, prepare the reaction system according to Table 1, and add samples according to the blank control, negative control, sample to be tested, positive Sexual control...