[Establishment of allele-specific diagnostic PCR method for identification of antlers].

OBJECTIVE

To establish a convenient, quick and accurate molecular method for the identification of crude drugs of antlers due to the difficult discrimination between the genuine antler and its adulterants.

METHOD

According to the alignment analysis of full length sequences of Cyth gene from closely relate species of Cervus, one pair of allele-specific diagnostic PCR primers was designed. Factors such as annealing temperature, dosage of polymerase, times of cycles and dosage of template DNA that influence the PCR results were also investigated.

RESULT

Based on the study mentioned above, about 323 bp positive band was amplified under the annealing temperature of 65 degrees C in the total volume of 25 microL PCR reaction using the genuine antler DNA as the template. Sequencing results proved that the positive band was the fragment of Cytb gene from both C. elaphus Linnaeus and C. nippon Temminck.

CONCLUSION

The established method, with higher specificity and reproducibility, could accurately differentiate genuine antler from its adulterants and would be widely used in Cervus related Chinese crude drugs' identification.