[Study on allele-specific diagnostic PCR of the traditional Chinese medicines of the deers].
OBJECTIVE
To develop a convenient and accurate method of DNA molecular marker for the identification of traditional Chinese medicines made of deers, consisting of pilose antler, penis and testis, tendon and foetus.


METHODS
Based on the analysis of DNA sequence of mitochondrial Cyt b gene from original animals of both genuine crude drugs, Cervus nippon and Cervus elaphus, and adulterants, a pair of allele-specific primers named as ILu01-L and ILu01-H were designed for distinguishing geniune crude drugs of deers from their adulterants.


RESULTS
The results of diagnostic PCR annealing at 64 degrees C for original animals showed that a 365 bp fragment was only amplified from DNA templates of Cervus nippon and Cervus elaphus. For the identification of medicinal materials total of 43 samples from 6 packages were tested under the same reaction conditions except for DNA templates extracted from these crude drugs. Only 9 samples mentioned above was shown to generate positive amplificon. The result indicate that of 8 samples from 1 package of pilose antler and only 1 sample of deer tendon was genuine crude drug. After that, 3 amplified fragments selected randomly were performed with sequencing analysis with the purpose of verifying the results from diagnostic PCR. Data from sequencing confirmed the reliability of diagnostic PCR identification.


CONCLUSIONS
The diagnostic primers designed in the present study were highly specific for Cervus nippon and Cervus elaphus, and they could be used for the authentication of traditional Chinese medicines made from the deer. The quality of the crude drugs of the deer in the current market is a problem and more effective quality control for these traditional Chinese medicines is urgently needed.
Liu
X H
XH
Institute of Genetic Resources, Nanjing Normal University, Nanjing 210097, China.
Wang
Y Q
YQ
Zhou
K Y
KY
Liu
Z Q
ZQ
Cao
L
L
D000483:Alleles; D000818:Animals; D004247:DNA / Q000032:analysis*; D003670:Deer / Q000145:classification / Q000235:genetics*; D004340:Drug Contamination; D005333:Fetus / Q000737:chemistry; D006733:Horns / Q000737:chemistry; D008297:Male; D008419:Materia Medica / Q000737:chemistry*; D010413:Penis / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D011786:Quality Control; D017422:Sequence Analysis, DNA
D008419:Materia Medica; D004247:DNA