SN/T 5197-2019Operation protocol of real-time quantitative PCR for bovine sarcoidosis virus (English PDF)

5.1 Main instruments and equipment

5.1.1 Fluorescence quantitative PCR instrument.

5.1.2 Low temperature high speed centrifuge, micro high speed centrifuge.

5.1.3 Low-temperature refrigerator.

5.1.4 Micro oscillator.

5.1.5 Biological safety cabinet.

5.1.6 Constant temperature water bath.

5.1.7 Autoclave.

5.1.8 Micro adjustable pipette.

5.2 Reagents and consumables

5.2.1 Nutrient solution. see A.1 for the preparation method.

5.2.2 Maintenance solution. see A.2.for the preparation method.

5.2.3 Cell dispersion. see A.3 for the preparation method.

5.2.4 Commercialized trace virus nucleic acid extraction kit or other commercialized kits.

5.2.5 Premix Ex Taq (Probe qPCR) fluorescent PCR buffer.

5.2.6 Primer. According to the gene sequence of the international standard strain of bovine nodular herpes virus (LSDV), design and synthesize in its most conservative ITR region sequence A pair of specific primers, F. 5'-TTGTCAGAAACGAGG-3', R. 5'-ATGCCTCACTTGTATTTGG-3' and a MGB the probe [(FAM)5'-TCTTGCTAAAATACCA-3'(MGB)] is prepared at 10 µmol/L and stored at -20°C.

Note. The reagents and water used above are all analytical reagents unless otherwise specified, and the water should meet the specifications of Grade 3 water specified in GB/T 6682.

5.3 Standard strains and cells

5.3.1 Standard strain. The Neethling strain, an international standard strain of LSDV, is used as the reference strain for the test.

5.3.2 Cells. Take healthy lamb testes according to conventional methods to prepare primary testicular cells; or use MDBK cells or Vero cells.

6.1 Positive control sample

Provided by the bovine nodular skin disease reference laboratory or prepared according to the following method. the bovine nodular eruption international standard strain (5.3.1) Inoculate primary testicular cells (5.3.2) at 10%, absorb at 37°C for 1 h, add maintenance solution (5.2.2), and incubate at 37°C with 5% CO2.

Harvest the virus suspension when the CPE reaches 70%. freeze and thaw 3 to 4 times, centrifuge at 5,000 r/min for 10 min, and take the supernatant for use. also may A plasmid containing the DNA sequence of the amplified region of LSDV ITR was used as a positive control.

6.2 Negative control sample

Provided by the bovine nodular skin disease reference laboratory or prepared according to the following method. freeze-thaw the well-growing primary testicular cells Centrifuge at 5,000 r/min for 3 to 4 times for 10 min, and take the supernatant for use.

7.1 Handling of samples

7.1.1 All experimental operations should meet the relevant requirements of GB 19489, GB/T 27401 and GB/T 27403.

Perform experiments in laboratories of grade and above.

7.1.2 Tissue samples. including skin, lungs, spleen, lymph nodes, muscles, etc. Take 2 g~5 g tissue sample and cut into small pieces, add Homogenize 5 mL~10 mL of pre-chilled PBS buffer for 2 minutes, make a suspension, freeze-thaw repeatedly 3 times, centrifuge at 4°C 5 000 r/min for 10 minutes, Take.200 µL of supernatant for nucleic acid extraction.

7.1.3 Blood sample. No special treatment is required, just take.200 µL for nucleic acid extraction.

7.2 Extraction and purification of viral DNA

Take.200 µL each of the negative and positive control samples and the samples processed in accordance with 7.1.2 and 7.1.3, and place them in a biological safety cabinet according to commercialization the operating instructions of the Mini Viral Nucleic Acid Extraction Kit require the extraction of viral DNA.

7.3 Fluorescence quantitative PCR detection

7.3.1 Fluorescence PCR reaction system

The fluorescence quantitative PCR reaction system for detecting bovine nodular herpes virus is shown in Table 1.Take bovine nodular herpes virus DNA as a positive control, with Beef tissue and primary testicular cell DNA without LSDV were used as a negative control, and water was used as a blank control.

Note. The volume of the fluorescent quantitative PCR reaction can be adjusted according to the actual situation.

7.3.2 Fluorescence PCR reaction parameters

Cycle parameters for PCR detection. 95 °C pre-denaturation 30 s, 95 °C 5 s, 60 °C 34 s, a total of 40 cycles, 60 °C 34 s collection Collect FAM fluorescence signal.

8.1 Result analysis

8.1.1 Read the test results. The principle of threshold setting is that the threshold line exceeds the highest point of the normal negative control amplification curve.

8.1.2 Different instruments can be adjusted according to the noise of the instrument.

8.2 Quality control standards

The test result of the negative control should have no specific amplification, and the Ct value of the positive control should be less than 28.0.

8.3 Judgment of results

8.3.1 Positive

The Ct value is less than or equal to 35, and there is an obvious amplification curve, it is determined that there is bovine nodular herpes virus nucleic acid in the sample.

8.3.2 Negative

If there is no specific amplification curve or the Ct value is greater than 40, it is determined that there is no bovine nodular herpes virus nucleic acid in the sample.

8.4 The principle of effective critical value judgment

35.0 Appendix A

(Normative appendix) Reagent preparation A.1 Nutrient solution DMEM medium plus 10% LSDV-free antibody calf serum, containing.200 IU/mL penicillin and.200 IU/mL streptomycin, Suction and sterilize.

A.2 Maintenance fluid DMEM medium plus 2% LSDV-free calf serum, containing.200 IU/mL penicillin and.200 IU/mL streptomycin, draw Filter out bacteria.

A.3 Cell dispersion

Pancreatin 2.50 g

Disodium ethylenediaminetetraacetic acid (EDTA) 0.20 g Calcium and magnesium-free PBS 1 000 mL Sterilize by suction filtration and store at -20°C for later use.