Specific PCR (Polymerase Chain Reaction) primers and method for authenticating Elaphodus cephalophus and application of specific PCR primers in authentication of Elaphodus cephalophus
A specific, hairy-crested deer technology, applied in biochemical equipment and methods, microbial determination/testing, DNA/RNA fragments, etc., can solve phenotypic plasticity and genetic variability, biological sex and developmental stage restrictions, and species that cannot be achieved Accurate identification and other problems, to achieve the effect of simple and easy identification, great promotion value, and reduce the difficulty of sampling
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Embodiment 1
[0029] The method for identifying the crested deer using specific PCR primers includes:
[0030] (1) Extract the total DNA of the sample to be tested:
[0031]Place 0.5 g of the sample to be tested numbered EC1 (see Table 1) in a centrifuge tube and cut the sample to be tested in the centrifuge tube with sterile scissors, then add 500 μL of lysate to the centrifuge tube successively, 30 μL of 10% sodium dodecyl sulfate (SDS) and 3 μL of 20 mg / ml proteinase K (PK), so that the concentration of SDS is 0.5%, and the final concentration of PK is 100 μg / ul, mix well and then digest in a water bath at 56 °C 12h until the liquid in the tube is clarified. Add 500 μL of balanced phenol to the above digested mixture, shake slightly for 5 minutes, and then place it in a centrifuge at 11,000 r / min for 10 minutes, and take the supernatant after centrifugation. Repeat the above centrifugation step twice. Add 500 μl of secondary mixture (chloroform: isoamyl alcohol = 24:1), shake slightly...
Embodiment 2
[0037] Operate according to the method of Example 1, the difference is that each 0.75 μL of a pair of PCR primers of 10pmol / L, the consumption of described total DNA is 45ng, the PCR amplification process comprises 28 cycles, and the denaturation temperature in a single cycle is 93 ℃, the annealing temperature is 55℃, the annealing time is 25s, and the extension temperature is 70℃. The amplified DNA was detected by electrophoresis on 1% EB-stained agarose gel, maintained at a voltage of 5V / cm for 30min, and observed on an ultraviolet gel imaging system. The electrophoresis result showed that a band appeared at 240bp.
Embodiment 3
[0039] Operate according to the method of Example 1, the difference is that each 1.5 μL of a pair of PCR primers of 10pmol / L, the consumption of described total DNA is 55ng, the PCR amplification process comprises 33 cycles, and the denaturation temperature in a single cycle is 95 ℃, the annealing temperature is 65℃, the annealing time is 40s, and the extension temperature is 73℃. The amplified DNA was detected by electrophoresis on 1% EB-stained agarose gel, maintained at a voltage of 5V / cm for 30min, and observed on an ultraviolet gel imaging system. The electrophoresis result showed that a band appeared at 240bp.
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