SN/T 5279-2020Technical specification for quarantine of eel herpes virus infection (English PDF)

7.1 Diagnosis of clinical symptoms

When HVA is infected, the clinical symptoms of eel are very different between groups and individuals. The common clinical symptoms are pectoral fins, the operculum and gill filaments are bleeding. Clinical symptoms can also be manifested as loosening and hyperplasia of mucus on the body surface, followed by shedding, and mucus defect plaques on the body surface. Follow as the disease progresses, the lesion reaches the dermis, ulcers occur, perforation and internal organs are exposed, the head is congested and red (red head), and the abdominal skin is bleeding.

Congestion of the gills, part of the fin rays and anus, rotten gills, protruding eyes and abdominal distension, swelling of the liver, discoloration or redness, swelling of the kidneys, swelling of the gallbladder, and intestine Inflammation, continued death, etc.

7.2 Sampling

The sampling quantity refers to the regulations of GB/T 18088 and SC/T 7103.According to the freshness of the sample, it can be oxygenated and refrigerated at 4 °C.

Or frozen and transported to the laboratory.

7.3 Sampling site

7.3.1 For fish with body length > 4 cm, gills, liver, spleen, kidneys and other parts can be taken for testing.

7.3.2 For fish with body length <= 4 cm, the whole fish can be rounded up for testing.

7.4 Tissue DNA extraction

7.4.1 Grind the tissue block into a homogenate shape with a grinding tool in an ice box, take about 25 mg of the tissue homogenate in a 1.5 mL centrifuge tube, and place it in a 1.5 mL centrifuge tube.

Add 500 µL of lysis buffer (see B.1) and proteinase K (see B.2) at a final concentration of 100 µg/mL. Place the above centrifuge tube Incubate in a 55 °C water bath for 1 h~2 h to fully lyse the tissue. Add 350 µL extract 1 (see B.3) and mix well 12 000 g Centrifuge for 5 min. Take the upper aqueous phase, add 350 µL extract 2 (see B.4), mix well and centrifuge at 12,000 g for 5 min. Take the upper water phase, Add 1.5 times the volume of absolute ethanol pre-cooled at -20 °C, mix well, place at room temperature for 10 minutes, centrifuge at 15,000 g for 10 minutes, and discard the supernatant. instant Centrifuge at time and carefully aspirate the remaining supernatant with a pipette. Leave it at room temperature for 5 minutes to dry the precipitate. Add 10 µL of sterilized double distilled water and flick the tube wall Dissolve the precipitate and use it as a PCR template.

7.4.2 Tissue DNA extraction can also use equivalent commercial DNA extraction kits and use them according to the instructions.

7.5 PCR detection and result judgment

7.5.1 PCR amplification system

10×PCR buffer (see B.5) 5 µL, 20 µmoL/L PCR primer (5.2) 1 µL each, 25 mmol/L MgCl2 5 µL, 10 mmoL/L dNTPs 1 µL, 5 U/µL Taq enzyme (5.1) 1 µL, DNA template 5 µL, add double distilled water to a total volume of 50 µL.

7.5.2 PCR amplification conditions

The reaction conditions are preheating at 94 °C for 5 min, then denaturation at 94 °C for 40 s, annealing at 65 °C for 45 s, and extension at 72 °C for 60 s, a total of 40 cycles.

Finally, extend at 72 °C for 10 min and store at 4 °C. The PCR reaction conditions can also be set according to the instructions of the commercial Taq enzyme.

7.5.3 Establish a comparison

Take the nucleic acid extract of the eel tissue sample that has been diagnosed as positive by PCR as a positive control template. Fetch has been diagnosed by PCR as Negative eel tissue sample nucleic acid extract was used as a negative control template. The blank control is sterile deionized water.

7.5.4 Agarose gel electrophoresis

Prepare a 1.5% agarose gel plate (containing 0.5 µg/mL EB) with TBE running buffer (see B.6), about 0.5 cm thick. will 6 µL of sample and 2 µL of loading buffer (see B.7) are mixed and added to the sample well, and a DNA standard Marker is set up as a control. 30 mA electricity Swim for about 0.5 h, and stop when the bromophenol blue indicator reaches the bottom of the gel plate. Observe the nucleic acid band under the gel imager.

7.5.5 Judgment of results

7.5.5.1 When a single nucleic acid band appears in the positive control at approximately 394 bp, and the negative control does not have the nucleic acid band, the experimental result Established.

7.5.5.2 If the sample has a nucleic acid band at about 394 bp, take the PCR amplified product and sequence it. The sequencing sequence is entered in Genbank.

Sequence homology is compared, and the homology is more than 95% can be judged as positive for HVA nucleic acid. Reference sequence Genbank serial accession number is.

7.5.5.3 Samples with no amplified bands, the size of the amplified bands are not 394 bp, and the homology between the sequencing result and the reference sequence is less than 95% can be judged Defined as HVA nucleic acid negative.

7.6 Fluorescent probe PCR detection and result judgment

7.6.1 Fluorescent probe PCR amplification system

7.6.2 PCR amplification conditions of fluorescent probes

The reaction conditions are 95 °C preheating for 10 min, then 95 °C denaturation for 15 s, 60 °C 30 s, 40 cycles, and collection at the end of each cycle FAM channel fluorescence signal.

7.6.3 Setting up a comparison

Set up negative control, positive control and blank control according to 7.5.3.

7.6.4 Judgment of results

7.6.4.1 The principle of threshold setting is that the threshold line just exceeds the highest point of the normal negative control amplification curve.

7.6.4.2 The blank control and negative control have no Ct value. The positive control has a typical positive amplification curve, and the Ct value is less than 30.

effective.

7.6.4.3 If the Ct value of the test sample is > 38, it is judged as negative for HVA nucleic acid. If the Ct value of the test sample is <= 35, and a typical amplification song appears Line, it is judged as positive for HVA nucleic acid. If the 35 < Ct value of the test sample <= 38, the result is judged to be suspicious, and it is necessary to repeat the fluorescence detection.

Needle PCR test, the result is still suspicious, it is judged as positive.