SN/T 5181-2020Technical specification for quarantine of hematopoietic organ necrosis of goldfish (English PDF)

9.1 PCR method

9.1.1 Establish a comparison

From the extraction of sample DNA, a positive control, a negative control, and a blank control need to be set up. Take the positives that are known to contain GFHNV Sex tissue was used as a positive control; healthy fish tissue was used as a negative control; an equal volume of sterile deionized water was used as a blank control.

9.1.2 Nucleic acid extraction (CTAB method)

Under aseptic conditions, homogenize the obtained tissue in a sterile mortar, and use pre-cooled phosphate buffer at a ratio of 1.10 (mass. volume) the tissue was resuspended, centrifuged at 8 000 r/min at 4°C for 5 min, and the supernatant was taken for inspection.

Take 450 µL of the tissue suspension into a 1.5 mL centrifuge tube, add 450 µL of CTAB solution, mix well, and incubate at 25°C for 2.5 h.

Add 600 µL extraction solution 1 to the centrifuge tube, shake vigorously for more than 30 s, and centrifuge at 12,000 r/min for 5 min. Take the upper water phase (Approximately 800 µL) to a new centrifuge tube, add 700 µL extraction solution 2, shake and mix for 30 s, and centrifuge at 12,000 r/min for 5 min. Be careful Take the upper water phase (approximately 600 µL) to a new centrifuge tube, add 1.5 times the volume of pre-cooled absolute ethanol (approximately 900 µL), and turn it upside down.

After pouring and mixing, the nucleic acid is precipitated at -20°C for more than 2 h. Centrifuge at 12 000 r/min for 30 min, and discard the supernatant. Place the centrifuge tube upside down Dry on water paper for 30 min at room temperature. Add 50 µL of TE buffer to the DNA precipitation and store at -20°C for later use.

A commercial nucleic acid extraction kit with the same effect can be used instead of the above method.

9.1.3 Reaction system

Add 5 µL of 10×PCR amplification buffer, 1 µL each of upstream and downstream primers (20 µmol/L), and dNTPs to the reaction tube (10 mmol/L) 1 µL, Taq enzyme (5 U/µL) 1 µL, add double distilled water to a total volume of 50 µL. After centrifuging the reaction tube momentarily, Place in the PCR machine.

9.1.4 Reaction conditions

94°C pre-denaturation 4 min; 94°C denaturation 1 min, 60°C annealing 1 min, 72°C extension 30 s, 40 cycles; 72°C Extend for another 10 minutes.

9.1.5 Agarose gel electrophoresis

Prepare 1.5% agarose (containing 0.5 µg/mL ethidium bromide) gel plate with TBE running buffer. Put it into the horizontal electrophoresis tank, Make the running buffer just cover the gel surface. Take 5 µL of PCR product and 1 µL of 6×loading buffer, mix well and add to the sample well, 5 V/cm After electrophoresis for about 30 minutes, place the gel on a gel imager to observe the results.

Other methods with the same effect can also be used, such as a capillary electrophoresis analyzer.

9.1.6 Sequencing

Perform gene sequence determination on PCR amplified products, and compare the sequence results with the reference sequence (see Appendix C) for homology Yes, or perform sequence homology analysis in Genbank, the homology should be greater than 98%.

9.1.7 Judgment of results

9.1.7.1 The determination of the results of the tested samples is based on the establishment of the positive and negative controls. Positive control can amplify one A specific band with a size of 366 bp. The negative control and blank control cannot amplify a 366 bp band.

9.1.7.2 If no band is amplified in the tested sample, or although there is a band but the size is not 366 bp, it is judged as the result of PCR detection.

Fruit negative. If a specific band with a size of 366 bp is amplified from the tested sample, and the sequencing result is consistent with the reference sequence, it is judged as the PCR method tested positive.

9.2 Real-time PCR method

9.2.1 Control establishment

See 9.1.1.

9.2.2 Nucleic acid extraction

See 9.1.2.

9.2.3 Reaction system

2×Real-time fluorescent PCR master mix 10 µL, upstream primer (10 µmol/L) 0.5 µL, downstream primer (10 µmol/L) 0.5...