SN/T 5186-2020Technical specifications for quarantine of abalone impetigo (English PDF)

5.1 Reagents

5.1.1 Unless otherwise specified, all chemical reagents required for the test should be of analytical grade.

5.1.2 The test water should meet the first-grade water specifications in GB/T 6682, and the test water used for DNA extraction and PCR test should be consumed.

Bacteria distilled water.

5.1.3 Culture medium.

5.1.4 Biochemical identification reagents. arginine dihydrogenase, lysine deshuttlease, ornithine deshuttlease, urease, sugar fermentation tube, trace biochemical Tube, biochemical identification reagent strip or kit, etc.

5.1.5 Bacterial genome extraction kit.

5.1.8 TBE running buffer.

5.1.9 Nucleic acid stains.

5.2 Equipment

5.2.1 Refrigerator.

5.2.2 Incubator.

5.2.3 Ordinary PCR machine.

5.2.4 High-speed refrigerated centrifuge.

5.2.5 Balance.

5.2.6 Fluorescent PCR machine.

5.2.7 Biological safety cabinet.

5.2.8 Electrophoresis instrument.

6.1 Sampling

The live abalone is dissected, and the tissues and organs such as foot, mantle, gonad, hepatopancreas, digestive tract, etc. are taken out and ground for use. Number of samples, Packaging, etc. can be implemented in accordance with GB/T 18088 or SC/T 7 103.If the importing country or region has specific requirements, press the importing country or region sampling.

6.2 Separation culture method

6.2.1 Enrichment

Biosafety should comply with the relevant regulations of GB 19489.

Add the collected abalone tissue to alkaline peptone water (see B.1) containing 3% NaCl, and incubate at 36 °C ± 1 °C 8 h~18 h.

6.2.2 Vaccination

Use an inoculating loop to inoculate the culture solution on the TCBS medium (see B.2), and incubate at 36 °C ± 1 °C for 18 h to 24 h.

6.2.3 Pure culture

Select a single yellow colony to inoculate the nutrient broth medium (see B.3), and culture it at 36 °C ± 1 °C for 18 h to 24 h.

6.2.4 Preliminary identification

6.2.4.1 Plate culture characteristics

After culturing at 36 °C ± 1 °C for 18 h to 24 h, Vibrio fluvialis will appear yellow colonies on TCBS.

6.2.4.2 Gram stain

After selecting the pure culture, perform gram staining on the suspicious colonies according to GB 4789.28.Vibrio fluvialis is a gram-negative bacterium.

6.2.5 Biochemical identification

The strains preliminarily identified as Vibrio fluvialis by 6.2.4 shall adopt an automatic biochemical identification system or be inoculated with arginine dihydrogenase, lysine and arginine dihydrogenase.

Bacterial microbiochemical identification tube for amino acid deshuttle enzyme, ornithine deshuttle enzyme, urease, sugar fermentation tube, oxidase, indole, etc., set at 36 °C ±1 °C Cultivate for 48 h, and judge according to the instructions of the bacterial microbiochemical identification tube.

Lysine deshuttlease, ornithine deshuttlease, urease and VP tests are all negative; lactose does not ferment, does not produce H2S, and glucose produces acid no gas is produced; samples that are both positive for arginine dihydrogenase and oxidase are judged to be positive for Vibrio fluvialis.

See Appendix C for the main biochemical identification results of Vibrio fluvialis.

6.3 PCR detection method

6.3.1 Laboratory requirements

Laboratory operations are carried out in accordance with the requirements of GB/T 19495.2.

6.3.2 Nucleic acid extraction

Take 100 µL of the culture solution cultured in 6.2.3 and place it in a centrifuge tube, place it in a boiling water bath at 100 °C for 10 min, centrifuge at 12,000 g for 3 min, and take the supernatant is used as a template for PCR detection, or stored at -20 °C for later use.

Commercial bacterial genome extraction kits can also be used.

6.3.3 PCR reaction system

A 25 µL reaction system was used to set up a positive control, a negative control and a blank control for the reaction. Sterilize the PCR reaction tube Double-distilled water 15.3 µL, template 1 µL, 3 mmol/L MgCl2 1.5 µL, 10×PCR buffer 2.5 µL, 0.2 mmol/L dNTP 0.5 µL,

5 U/µL Taq enzyme 0.2 µL, 2.5 mmol/L primer F1 and primer R1 2 µL each.

6.3.4 PCR reaction conditions

94 °C pre-denaturation 4 min; 94 °C denaturation 1 min, 56 °C annealing 1 min, 72 °C extension 2 min, 30 cycles; 72 °C extension 7 min; keep warm at 4 °C.

6.3.5 Electrophoresis

Prepare a 1% agarose plate with TBE running buffer (see B.4) and add nucleic acid EB stain (see B.5). Add 8 µL PCR Mix the product with 2 µL of sample buffer (see B.6) and add it to the gel wells, and run at 5 V/cm for about 30 minutes to establish a DNA molecular weight standard as a control, observe the results under an ultraviolet lamp or in a gel imager.

6.3.6 Determination of PCR results

The positive control showed a band at 332 bp, and the negative control and blank control did not show a band, so the experiment was established.

The sample showed no band at 332 bp and was judged as negative. The sample has a band at 332 bp, which should be cut and recovered for DNA detection.

See Appendix D for the reference sequence.

6.4 Real-time fluorescent PCR detection method

6.4.1 Nucleic acid extraction

See 6.3.2 Operation.

6.4.2 Fluorescence PCR reaction system

A 25 µL reaction system was used to set up a positive control and a negative control for the reaction. Add 8.4 µL of sterilized double-distilled water to the PCR reaction tube, Fluorescence PCR reaction mixture 12.5 µL, 10 µmol/L primer F2 and primer R2 each 0.3 µL, 10 µmol/L probe 0.5 µL, Template 3 µL. The reaction system can also be prepared before sample addition. If there are n samples, n 2 reaction systems can be prepared and then dispensed in each reaction system.

Should be managed.

6.4.3 Fluorescence PCR reaction conditions

The first stage. 95 °C 10 min, 1 cycle; the second stage. 95 °C 15 s, 60 °C 1 min, 40 cycles, 60 °C Collect fluorescence during annealing extension. The reporter fluorescence is set to FAM, the quenching fluorescence is set to TAMRA, and the reference fluorescence is set to None (multiple fluorescence ECLIPSE is optional for PCR quenching fluorescence).

6.4.4 Judgment of fluorescent PCR results

The negative control has no Ct value or Ct value >= 40, and the positive control has a Ct value