特异性PCR方法鉴别鳖甲药材和饮片

Specific PCR method for identification of Trionycis Carapax and its preparation

鳖甲为临床常用的动物药材,炮制加工后鳖甲药材及饮片形态特征丢失,难以鉴别。为建立高效稳定的鳖甲DNA鉴别方法,该研究将SDS法与柱纯化相结合,使用通用引物进行PCR扩增和测序,并根据鳖甲及其混伪品序列差异,寻找特异性SNP位点设计了鉴别引物,对PCR反应条件进行优化,并进行适用性及准确性考察。当退火温度为62℃,循环次数为35,使用设计的鳖甲特异性鉴别引物Biejia-272.F/R进行PCR扩增,鳖甲药材、饮片及醋鳖甲均扩增获得约300 bp的特异性鉴别条带,混伪品无条带。该方法可作为一种快速准确鉴别鳖甲正伪品的鉴定方法。

Trionycis Carapax is a commonly used animal medicine in Chinese medicine. It′s difficult to identify Trionycis Carapax and its adulterants because of the loss of morphological characteristics after processing. To establish an efficient and stable method to identification Trionycis Carapax, this study combines SDS method with column purification to extract genomic DNA, uses universal primers for polymerase chain reaction(PCR) amplification and sequencing, and designs the specific primers based on the differences in the sequences of Pelodiscus sinensis and their adulterants. When the annealing temperature was 62 ℃ and the number of cycles was 35, the designed primer Biejia-272.F/R was used for PCR amplification and got optimum results. The crude drug and preparation of P. sinensis were all amplified to obtain a specific band of approximately 300 bp, while the adulterants showed no such a band. This method can be used as a rapid and accurate method to identify the authenticate of P. sinensis.