SN/T 5202-2020Technical Specification for Sika Deer Species Identification - Real-time Fluorescent PCR Method (English PDF)

5.1 Primers and probes for detection

Refer to Appendix A for the reference sequence of sika deer gene expansion target.

5.2 Reagents

5.2.1 CTAB extraction buffer. 20 ng/L CTAB, 0.1mol/L Tris-HCl, 1.4 mol/L NaCl, 0.02 mol/L EDTA, pH 8.0

5.2.2 CTAB precipitation solution. 5 g/L CTAB, 40 mmol/L NaCl

5.2.3 Proteinase K solution (20 mg/mL)

5.2.4 Trichloromethane

5.2.5 Isopropanol

5.2.6 70% ethanol

5.2.7 TE buffer. 10 mmol/L Tris-HC (pH 8.0), 1 mmol/LEDTA (pH 8.0).

5.2.8 Fluorescence quantitative PCR master mix. 10 µL The reaction system includes. 1 U~2 U Taq enzyme, 1×PCR buffer, 2.5 mmol/L~4.0 mmol/L Mg2+, 0.2 U~1 U UNG enzyme, 0.2 mmol/L d(A, C, G)TPs, 0.2 mmol/L~0.4 mmol/L dUTP, 400 nmol/L ROX dye (some fluorescent PCR machines do not require ROX calibration). If there is an equivalent commercial kit, then these equivalent products can be used.

6.1 Real-time fluorescent PCR instrument

6.2 Centrifuge (centrifugal force 12 000 g)

6.3 Centrifuge tube (1.5 mL, 2.0 mL)

6.4 Nucleic acid protein analyzer or UV spectrophotometer

6.5 Micropipette (2.5 µL, 20 µL,.200 µL, 1 000 µL)

6.6 Mortar or crushing device.

6.7 Constant temperature water bath (10 °C~90 °C)

6.8 Balance (sensitivity 0.01 g)

7.1 DNA extraction of samples

Weigh.200 mg or.200 µL of the ground sample into a 2.0 mL centrifuge tube, and add 1.5 mL CTAB extraction buffer and 10 µL of proteinase K solution, 65°C for 30 min, shake and mix from time to time; centrifuge at 13,000 g for 10 min, transfer the supernatant to a clean in the centrifuge tube, add 750 µL of chloroform/isoamyl alcohol (24.1, V/V), mix well, centrifuge at 13,000 g for 5 min, and transfer the supernatant to in a clean centrifuge tube; add 2 times the volume of CTAB precipitation solution, stand still at room temperature for 60 minutes, centrifuge at 13,000 g for 15 minutes, and discard the supernatant. Join 350 µL of NaCl solution will resuspend the pellet. Then add 350 µL of chloroform, vortex to mix, and centrifuge at 13,000 g for 10 min.

After transferring the supernatant, add 0.6 times the volume of isopropanol to precipitate the nucleic acid, leave it at room temperature for 20 minutes, centrifuge at 13,000 g for 10 minutes, and discard the supernatant.

Add 500 µL of 70% ethanol to wash once and dry; add 100 µL of TE to dissolve the precipitate. Use immediately or store at -20°C for later use.

An equivalent DNA extraction method can also be used to extract the DNA template.

7.2 Real-time fluorescent PCR detection

7.2.1 Amplification reaction system.

The volume is 20 µL. 2× fluorescence quantitative PCR master mix 10 µL, primer pair (10 nmol/L) each 1.0 µL, template DNA (100 ± 50 ng DNA) 2.5 µL, fluorescent probe (10 nmol/L) 0.5 µL, ddH2O 5.0 µL.

7.2.2 Reaction conditions.

50 °C, 2 min; 95 °C, 10 min; 95 °C, 15 s, 60 °C, 1 min, 40 cycles. The reaction cycle parameters can be based on Make appropriate adjustments due to the different models of the thermal cycler.

7.2.2 Setting of control in the reaction system

In the detection process, a positive control, a negative control and a blank control were set up respectively. Use samples known to contain sika deer or sika deer-derived ingredients as Positive control, use a sample known to not contain sika deer-derived components as a negative control, and use an equal volume of ddH2O instead of template DNA as a blank Contrast.