SN/T 5188-2020Technical specifications for quarantine of Edwards sluggish disease (English PDF)

5.1 Instrument and equipment

Microscope. 10×~100×.

5.2 Medium

5.2.1 Blood agar. see B.1.

5.2.2 Nutrient agar. see B.2.

5.2.3 Ferrous sulfate agar. see B.3.

5.2.4 Semi-solid agar. see B.4.

5.2.5 Sodium malonate medium. see B.5.

5.2.6 Peptone water. see B.6.

5.2.7 Glucose peptone culture medium. see B.7.

5.2.8 Lysine decarboxylase culture medium. see B.8.

5.2.9 ONPG medium. see B.9.

5.2.10 Sugar fermentation tube. see B.10.

5.2.11 Commercial media can be used.

5.3 Collection and treatment of disease materials

Aseptically take an appropriate amount of liver, kidney and diseased tissues of diseased animals, place them in a sterile centrifuge tube, rinse with sterile saline, and homogenize for later use. Such as the test cannot be performed immediately, and the collected or processed samples must be stored at 4 °C ~ 8 °C, and no more than 24 hours.

5.4 Separation culture

Take samples and homogenize them, inoculate blood agar plates with streaks, and incubate at 28 °C ± 1 °C for 24 h to 48 h. Pick out gray, round, moist, the translucent single colony was purified and cultured on a common nutrient agar plate.

5.5 Staining microscopic examination

The suspicious bacteria were subjected to Gram stain and observed under a microscope. Edwards tarda is Gram-negative Brevibacterium, no capsule, no the spores are approximately (0.5 µm ~ 1 µm) × (1 µm ~ 3 µm) in size.

5.6 Biochemical test

5.6.1 Oxidase test

Take the white clean filter paper and dip it into the colony, add a drop of 1% dimethyl p-phenylenediamine hydrochloride solution, the positive ones will appear pink, and gradually add Deep; add a drop of 1% alpha-naphthol solution, the positive person will appear bright blue within half a minute. The negative person does not change color within two minutes. Or mao the reagent is sucked by a thin pipette and directly dripped onto the colony. The color reaction is the same as the above.

5.6.2 Biochemical identification test

Suspicious bacteria with negative oxidase test shall be biochemically identified according to Appendix C.

6.1 Reagents and materials

6.1.1 Water

Should comply with GB/T 6682 Grade 1 water specifications.

6.1.2 DNA extraction solution

40 mmoL/L Tris-HCl, pH8.0 20 mmol/L sodium acetate, 1 mmol/L EDTA, 1% SDS.

6.1.3 10×PCR buffer

Potassium chloride (KCl). 500 mmol/L; Tris HCl (pH 8.3). 100 mmol/L; gelatin. 0.1%; MgCl2.15 mmol/L.

6.1.4 DNTPS

dATP, dGTP, dTTP, dCTP each 2.5 mmol/L.

6.1.5 Taq DNA Polymerase (5 U/µl)

6.1.6 50×TAE buffer

Weigh 484 g Tris, measure 114.2 mL glacial acetic acid,.200 mL 0.5 mol/L EDTA (pH 8.0), dissolve in water, and dilute to 2 L.

After aliquoting, autoclave for later use.

6.1.7 DNA Marker

6.1.8 SYBR Green I nucleic acid dye

6.2 Apparatus and equipment

6.2.1 Thermal cycler.

6.2.2 Electrophoresis device.

6.2.3 Gel imaging system.

6.2.4 PCR ultra-clean workbench.

6.2.5 High-speed desktop centrifuge (speed above 12 000 r/min).

6.2.6 Bench-top centrifuge (speed above 2 000 r/min).

6.2.7 Micro adjustable pipette (2 µL, 10 µL, 100 µL, 1 000 µL).

6.2.8 Fluorescence quantitative PCR instrument.

6.3 Template extraction

6.3.1 Boiling extraction method

Take 1 mL of the sample to be tested (enrichment culture solution or isolated bacterial colony suspension), add it to a 1.5 mL centrifuge tube, and centrifuge at 8 000 r/min 4 min, aspirate the supernatant, add 50 µL DNA extract, mix well, boil water bath for 5 min, centrifuge at 12 000 r/min for 5 min, take the supernatant the solution is stored at -20 °C for later use, and it can be stored at -70 °C for a long time.

6.3.2 Reagent extraction method

Take 1 mL of the sample to be tested (enrichment culture solution or isolated bacterial colony suspension), add it to a 1.5 mL centrifuge tube, and centrifuge at 8 000 r/min 4 min, aspirate the supernatant, add.200 µL DNA extraction solution, mix thoroughly, and centrifuge at 12,000 r/min for 10 min. Transfer the supernatant Add an equal volume of Tris saturated phenol to a new centrifuge tube, mix well, and centrifuge at 12,000 r/min for 3 min. Take the water layer, add an equal volume of chloroform, Mix well and centrifuge at 12 000 r/min for 3 min. Take out the supernatant and precipitate it with pre-cooled 2 times volume of absolute ethanol, centrifuge at 15 000 r/min for 15 min, Discard the supernatant. Wash twice with 400 µL 70% ethanol. After vacuum drying, dissolve the DNA in 50 µL TE or ultrapure water, and ice at -20 °C Store in the box for later use, and can be stored for a long time at -70 °C.

6.3.3 Kit method

Use an equivalent commercial bacterial DNA extraction kit and follow the instructions to extract and prepare template DNA.

6.4 Polymerase Chain Reaction (PCR)

6.4.1 Primer

6.4.2 Test control

The blank control uses water instead of the DNA template; the negative control uses the DNA of non-target bacteria as the template for the PCR reaction; the positive control uses Use the DNA (or plasmid) containing the detection sequence as the template for the PCR reaction.

6.4.4 PCR reaction

Put the prepared PCR tube into the PCR reaction instrument. See Table 1 for the setting of cycling conditions.

6.4.5 Electrophoresis

Prepare 1% ~ 2% agarose gel with running buffer (1×TAE) (add ethidium bromide at 55 °C~60 °C to the final concentration 0.5 µg/mL, can also be stained after electrophoresis). Take 5 µL ~ 10 µL PCR amplification products and mix them with 2 µL loading buffer respectively, Do spotting and use DNA Marker as a reference. 3 V/cm ~ 5 V/cm constant pressure, electrophoresis for 20 min ~ 40 min, use gel for electrophoresis results the imaging analysis system records and saves, and can also observe the electrophoresis results under 365 nm wavelength ultraviolet light.

6.4.6 Quality control

When one of the following conditions is not met, the experiment is deemed invalid.

a) Blank control. no amplified bands.

b) Negative control. no amplified band

c) Positive control. there is an amplified band with the same size as expected.

6.4.7 Judgment of results

If there is no amplified band of the corresponding size in the sample to be tested, the PCR test result of the sample can be reported as negative.

If there is an amplified band of the corresponding size in the sample to be tested, the PCR test result of the sample can be reported as positive.

For a positive result, the PCR product can be recovered and sequenced, and the sequencing result can be compared with the reference sequence (see Appendix D), or the test can be the sequence results are submitted to the NCBI website for BLAST analysis.

6.5 Real-time fluorescent PCR

6.5.1 Primers and probes

6.5.2 Test control

The blank control is set to replace the DNA template with water; the negative control uses non-target bacteria...