SN/T 5194-2020Technical specifications for quarantine of sheep infectious pustular dermatitis (English PDF)

5.6 Primers and probes

A set of specific primers were synthesized based on the specific genes of sheep infectious pustular dermatitis. The size of the amplified product is 488 bp.

5.7 Positive control. a standard strain that can be traced to the source, or DNA containing the target fragment can also be used.

5.8 Molecular biology reagents. 10×PCR buffer, dNTP (2.5 mmol/L), Taq DNA polymerase (5U/µL), ExTaq Hs (5U/µL).

5.9 0.2 mL and 1.5 mL plastic centrifuge tubes.

5.10 Tips, used with pipettes.

5.11 Sterilized Erlenmeyer Flask. 250 mL, 500 mL.

7.1 Clinical symptoms

The clinical symptoms are mainly divided into lip type, hoof type and vulva type, see Appendix B in B.1.

7.2 Necropsy changes

The main lesions are shown in Appendix B in B.2.

7.3 Epidemiology

The main epidemiology is shown in Appendix B, B.3.

7.4 Initial diagnosis

Make a preliminary diagnosis based on clinical symptoms, changes in necropsy, and epidemiology.

8.1 Sample processing

Collect diseased materials such as pustules on the skin of suspected sheep scabs, add 0.01 mol/L (pH7.2) PBS and grind to make 1.10 (mass.volume) the suspension was centrifuged at 5 000 r/min for 30 min, the supernatant was taken, and the double antibody (penicillin, streptomycin) at a concentration of 10 000 IU/mL was added at a ratio of 20%.

After feeling overnight at 4 °C, store it for later use.

8.2 Sample inoculation and culture

After the primary cells of calf testis have grown up to a 25 cm2 cell culture flask monolayer, discard the supernatant nutrient solution, and inoculate 1 mL of diseased material to treat the supernatant.

Adsorbed at 37 °C for 2 h, discard the supernatant and add MEM cell maintenance solution containing 2% serum, culture at 37 °C statically, and set up a normal cell pair Photograph and control infected cells. Observe cell growth and cytopathic (CPE) every day. If the cells did not emerge within 4 to 5 days after inoculation the current disease will be blindly transmitted for 3 consecutive generations and then the culture will be collected; if CPE appears, the culture will be collected directly.

8.3 Result identification

8.3.1 The control cells are normal, and the cells inoculated with the sample have no CPE after blind transmission for 3 generations, and they are judged as negative.

8.3.2 Small lesions appear at the initial stage of the cells, and the cells in the lesions are rounded and refracted. Further culture, CPE increased, and all cells shed their walls.

After continuous passage, complete CPE can be produced after 24 h to 48 h.

8.3.3 The cultures with the above-mentioned typical CPE should be further identified by PCR and fluorescent PCR.

9.1 Preparation of template DNA

Take 465 µL of 8.1 supernatant of the sample to be tested, add 25 µL of 10% sodium lauryl sulfate and 10 µL of 20 mg/mL proteinase K, Place on a 50 °C water bath shaker for 2 h; add the same amount of saturated phenol solution 500 µL, shake and mix for 20 s, centrifuge at 12,000 r/min for 5 min, and take Supernatant; add an equal volume of phenol. chloroform. isoamyl alcohol (25.24.1), shake and mix for 20 s, centrifuge at 12,000 r/min for 5 min, Take the supernatant; add an equal volume of chloroform. isoamyl alcohol (24.1), shake and mix for 20 s, centrifuge at 12,000 r/min for 5 min, and take Supernatant; finally add twice the volume of absolute ethanol, mix upside down, centrifuge at 12,000 r/min for 10 minutes, discard the supernatant, and leave at room temperature After drying, add 50 µL of double distilled water to dissolve the precipitate, and store the obtained DNA template at -20 °C for later use. In addition, DNA extraction can also be used Use an equivalent commercial DNA extraction kit and follow the instructions.

9.2 Reaction system

The reaction system is 25 µL. 10×PCR buffer (Mg2 Plus) 2.5 µL, dNTP (2.5 mmol/L) 2.0 µL, primers (25 µmol/L) each 1 µL, Taq DNA polymerase (5U/µL) 5 µL, template DNA 1 µL, and ddH2O make up 25 µL.

9.3 Reaction conditions

Pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 40 s, 40 cycles; extension at 72 °C Stretch for 10 min and store at 4 °C.

9.4 Observation of results

Prepare a 1% agarose gel with 1×TAE running buffer, and add ethidium bromide at a final concentration of 1 µg/mL. Use 100 bp DNA ladder Marker was used as a control for electrophoresis of PCR products. Observe the specificity of the target size on the gel imaging system Bands. If necessary, the amplified bands can be sequenced and analyzed.

9.5 Judgment of results

There was no specific band amplification in the blank control and the negative control, and the positive control showed a band of 488 bp (see Appendix C).

Bottom. The target band in the sample to be tested is judged to be a positive result of nucleic acid; the sample to be tested without the target band is judged to be negative.

10.1 Preparation of template DNA

Same as 9.1.

9.2 Reaction system

The reaction system is 25 µL. 10×PCR buffer (Mg2 Plus) 2.5 µL, dNTP (2.5 mmol/L) 2.0 µL, primers (25 µmol/L) 1 µL each; ExTaq Hs (5 U/µL) 5 µL, template DNA 1 µL, probe 1 µL, and ddH2O make up 25 µL.

10.3 Reaction conditions

Pre-denaturation at 95 °C for 10 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s, and extension at 72 °C for 40 s, where fluorescence is collected for 40 cycles cycle.

10.4 Judgment of results

10.4.1 Quality control standards

The negative control and blank control have no Ct value and no amplification curve; the Ct value of the positive control should be less than or equal to 32, and typical amplification Increasing curve; if the negative control, blank control and positive control do not meet the above conditions, this experiment will be considered invalid.

10.4.2 Result description and judgment

10.4.2.1 Negative

No Ct value and no amplification curve, indicating that there is no sheep infectious pustular dermatitis virus nucleic acid in the sample.

10.4.2.2 Positive

The Ct value is less than or equal to 32, and a typical amplification curve appears, indicating that there is sheep infectious pustular dermatitis virus nucleic acid in the sample.

Appendix A

(Normative)

Cell and reagent preparation A.1 Preparation of bovine testis primary cells Collect the testicles of a 1-day-old calf, spray the outer coat of the testicles with 75% alcohol, and take it out with sterile tweezers after 1 min to 2 min the bovine testicles were placed in another sterile dish, and the testicles were washed 3 times with PBS containing 100 units of double antibody. Ophthalmological scissors cut to wrap the outside of the testis of the connective tissue, peel off and remove the substance...