SN/T 1467-2020Technical specifications for gosling plague quarantine (English PDF)

5.1 Sample collection

5.1.1 For dead or dying animals, samples of liver, pancreas, spleen, kidney, brain, etc. shall be taken aseptically. For live animals, collect whole blood. For animal products, Collect internal organs and tissues.

5.1.2 Sterile equipment should be used for the collection and packaging of samples, and should be sealed, refrigerated and transported to the laboratory immediately after collection.

5.1.3 The sample is made into 10% to 20% suspension with phosphate buffered saline (PBS) containing antibiotics at pH 7.0 ~ 7.4 (see Appendix C for the preparation method).

The floating liquid was centrifuged at 4 °C 3 000 r/min for 10 min, and the supernatant was filtered and sterilized through a 0.22 µm filter membrane. Samples collected or processed at 2 °C ~ Store at 8 °C for no more than 4 days. For long-term storage, it should be placed in a -70 °C refrigerator, but repeated freezing and thawing should be avoided.

5.2 Virus isolation test

5.2.1 Reagents

- Penicillin (working concentration 10 000 IU/mL);

--Streptomycin (working concentration 10 000 IU/mL);

- 9-day-old ~11-day-old SPF goose embryo.

5.2.2 Main equipment

--Biological safety cabinet;

- Centrifuge;

-Constant temperature incubator;

--Micro adjustable pipette.

5.2.3 Test procedure

5.2.3.1 Goose embryo inoculation

Aspirate the filtered and sterilized sample treatment solution, and inoculate at least 5 SPF goose embryos aged 9 to 11 days through the allantoic cavity, 0.2 mL/piece, Incubate at 35 °C ~37 °C for 4 d to 7 d. Check the growth of goose embryos every day after inoculation.

5.2.3.2 Virus harvest

The dead embryos after 18 h and the surviving goose embryos at the end of the culture were collected and placed in a refrigerator at 4 °C for 4 h to 24 h, and the allantoic fluid was collected aseptically.

5.2.4 Virus identification

Use agar diffusion test or polymerase chain reaction (PCR) for gosling plague virus identification.

5.3 Agar immunodiffusion test

5.3.1 Reagents and materials

5.3.1.1 Unless otherwise specified, all reagents are analytical reagents or biochemical reagents.

5.3.1.2 Agar plate (see Appendix C for the preparation method).

5.3.1.3 Gosling plague virus positive serum (provided by the designated unit).

5.3.1.4 Gosling plague virus negative serum (provided by the designated unit).

5.3.1.5 Gosling plague virus standard antigen (provided by the designated unit).

5.3.2 Main equipment

Micro adjustable pipette, alcohol lamp, 37 °C thermostat.

5.3.3 Detection antibody

5.3.3.1 Preparation of tested serum

Blood was collected to separate serum according to conventional methods. There should be no hemolysis and no bacterial contamination. Store at 4 °C.

5.3.3.2 Treatment of the yolk of the inspected goose

For the treatment of the tested goose egg yolk, use a syringe (without needle) to draw 20 mL of fresh goose egg yolk into a centrifuge tube, and add the same amount of physiological salt Shake well with water, add 20 mL of chloroform and shake for 30 minutes, centrifuge at 3 500 r/min for 30 minutes, and take the supernatant (Approximately 20 mL) into a dialysis bag, put in 40% polyethylene glycol and concentrate (approximately 6 h~12 h) to 1 mL~2 mL, aspirate the concentrated solution and use Wash the dialysis bag with 1 mL ~ 2 mL of normal saline, aspirate and combine in the concentrated solution, and control the final concentration to 5 times. Finally at 3 500 r/min Centrifuge for 30 minutes, add 0.01% thimerosal to the supernatant, and freeze and store (-20°C) for inspection.

5.3.3.3 Punch

The reaction hole is used and punched now. On the prepared agar plate, punch holes according to Figure 1, with a hole diameter of about 5 mm and a hole distance of 2 mm~5 mm. Put in the hole Use a needle to gently pick out or aspirate the agar, do not damage the edge to avoid leakage.

figure 1

5.3.3.4 Back cover

Use an alcohol lamp to lightly bake the bottom of the plate or glass plate, and close the bottom of the hole to prevent side leakage.

5.3.3.5 Dosing

Use a micropipette to suck the antigen suspension and add it to well G, add the positive serum control to the well, and press the test serum or egg yolk supernatant Add them to the remaining holes according to the order, and each hole should be filled with no overflow. Change a tip for each sample added.

5.3.3.6 Function

After adding the sample, let it stand for 10 minutes, then put the plate upside down horizontally or put the glass plate upright in the wet box, and put it in the wet box at room temperature Act at 22 °C ~ 25 °C for 48 h to 72 h, observe and record the results at 24 h, 48 h and 72 h respectively.

5.3.3.7 Judgment of results

5.3.3.7.1 Under a dark background, place the agar plate under a fluorescent lamp or under strong side light to observe. If there is a line between the standard positive serum and the antigen well Clear and dense white precipitation line, the test is established, otherwise it is deemed invalid and needs to be redone.

5.3.3.7.2 If a precipitation line appears between the tested serum well and the central antigen well, and it coincides with the end of the precipitation line of the standard positive serum, then the tested serum was judged to be positive (shown in Figure 1 for hole 1).

5.3.3.7.3 If there is no precipitation line between the tested serum well and the central antigen well, but one end of the standard positive serum precipitation line is bent toward the tested blood Clear the hole (shown in Figure 1 as well as No. 3), then the tested serum in this hole is judged to be weakly positive (for those who are weakly positive, the test should be repeated, and for those who are still not weakly positive, Judged as positive).

5.3.3.7.4 If there is no precipitation line between the tested serum well and the central antigen well and the precipitation line of the standard positive serum is straight to the tested serum well or those who deviate to the outside (as shown in the No. 2 hole in Figure 1) are judged as negative by the test serum.

5.3.3.7.5 If the precipitation line between the tested serum well and the central antigen well crosses the precipitation line between the standard positive serum and the antigen well Straight extension (shown in No. 4 hole in Figure 1), it is judged as a non-specific reaction, and the test should be repeated. If non-specific reaction still occurs, judge is negative.

5.3.4 Detection of antigen

5.3.4.1 Antigen preparation

Take out the chorioallantoic membrane from the goose embryo, wash it with pH7.2, 0.01 mol/L PBS, grind it with a grinder, and freeze-thaw for 3 consecutive times~ Centrifuge for 4 times at 1,000 r/min for 10 minutes, take the supernatant, and add the formaldehyde solution at a final concentration of 0.1%.

5.3.4.2 Punch

On the prepared agar plate, punch holes according to Figure 2 with a hole diameter of about 5 mm and a hole distance of 2 mm ~ 5 mm. Gently apply the agar in the hole with a needle Pick out or suck it out without hurting the edges to avoid leakage.

figure 2

5.3.4.3 Back cover

Use an alcohol lamp to lightly bake the bottom of the plate or glass plate, and close the bottom of the hole to prevent side leakage.

5.3.4.4 Dosing

Use a micropipette to draw the standard positive serum and add it to well S, drop the standard antigen into the well, and prepare the antigen in accordance with 5.3.4.1 the suspension is added to other peripheral holes, and each hole is filled with no overflow. Change a tip for each sample added.

5.3.4.5 Function

After the sample is added, let it stand for 5 min ~ 10 min, and then put the plate upside down horizontally or the glass plate horizontally upright in the wet box, at 37 °C in the incubator, observe and record the results after 24 h, 48 h and 72 h respectively.

5.3.4.6 Judgment of results

5.3.4.6.1 Under a dark background, place the agar plate under a fluorescent lamp or under strong side light for observation. If the standard antigen suspension wells and the central standard positive serum If a clear and dense white precipitation line appears between the holes, the test is valid, otherwise it is deemed invalid and needs to be repeated.

5.3.4.6.2 If there is a precipitation line between the tested antigen solution and the central standard positive serum well, and it is in phase with the end of the precipitation line of the standard antigen suspension.

If it is matched, it can be determined that the tested antigen suspension is positive (shown in Figure 2 for hole 1).

5.3.4.6.3 If there is no sedimentation line between the tested antigen and the central standard positive serum well, but the sedimentation line of the standard antigen suspension is bent toward If the antigen suspension hole (No. 3 hole as shown in Figure 2), the tested antigen suspension is judged to be weakly positive (for those who are weakly positive, the test should be repeated, and the test is still not weak.

Sex is judged as positive).

5.3.4.6.4 If there is no precipitation line between the test antigen suspension and the central standard positive serum well and the standard antigen suspension precipitation line is straight to the test the antigen suspension hole or the person who is bent to the outside (shown in Figure 2 as No. 2 hole), the tested antigen suspension is judged as negative.

5.3.4.6.5 If the precipitation line between the tested antigen suspension and the central standard positive serum is the same as the standard antigen suspension and the central standard positive serum well If the precipitation line between them crosses and straightens (as shown in Figure 2 for hole 4), it is judged as a non-specific reaction, and the test should be repeated. If non-special Heterosexual reaction is judged as negative.

5.4 Polymerase chain reaction (PCR)

5.4.1 Reagents

5.4.1.1 Unless otherwise specified, all reagents are analytical reagents or biochemical reagents.

5.4.1.2 Experimental water (should meet the specifications of the first grade water in GB/T 6682), Taq enzyme, PCR buffer (matched with Taq enzyme), chlorine Magnesium (MgCl2, 25 mmol/L), dNTPs (dATP, dCTP, dGTP, dTTP, 2.5 mmol/L each), phenol/chloroform/isoamyl Alcohol (volume ratio 25.24.1), chloroform, isopropanol (pre-cooled at -20 °C), agarose, DNA relative molecular weight standard Marker DL 2 000.

5.4.1.3 Physiological saline, DNA extraction solution, 75% ethanol, TE solution (pH 8.0), electrophoresis buffer (1×TBE or 1×TAE), Ethidium bromide solution (10 mg/mL), loading buffer, see Appendix C for the preparation method.

5.4.1.4 Gosling plague standard strain or its DNA (provided by the designated organization).

5.4.1.5 Primer.

5.4.2 Equipment

- PCR thermal cycler,

--Biological safety cabinet,

--Gel imaging system,

--Disinfection and sterilization pot,

--Ice maker,

--Nucleic acid protein analyzer,

--High-speed refrigerated centrifuge,

--Desktop small centrifuge,-Low temperature refrigerator,

--Ultra pure water device,-Vortex oscillator.

5.4.3 Sample preparation

After adding a small amount of normal saline to solid samples such as tissues, they are fully homogenized to prepare a 10%-20% suspension for use; liquid samples are filled Take it directly after mixing.

5.4.4 Preparation of template DNA

5.4.4.1 Preparation of sample template DNA

Take.200 µL of the sample suspension prepared above, add 750 µL of DNA extract, incubate at 65 °C for 30 min, add phenol/trichloromethane 500 µL alkane/isoamyl alcohol, shake and mix well, centrifuge at 13,000 r/min for 5 min, aspirate the supernatant and add an equal volume of chloroform, shake and mix Centrifuge at 13 000 r/min for 5 min, draw 500 µL of the supernatant and mix well with 400 µL of isopropanol, and centrifuge at 13 000 r/min Rinse the precipitate with 75% ethanol for 5 minutes, centrifuge at 13,000 r/min for 5 minutes, discard the supernatant, and dissolve the precipitate in 30 µL TE solution after drying it is used immediately for testing or stored at -20 °C for a short period of time.

Other proven DNA extraction methods or equivalent commercial viral DNA extraction kits can also be used, and follow their instructions operating.

5.4.4.2 Control establishment

In the detection process, a positive control, a negative control and a blank control are set respectively. The positive control is a standard strain of gosling plague...