猪胆粉及其中成药的特异性PCR鉴别方法

目的:建立一种适用于猪胆粉及其中成药的特异性聚合酶链式反应(PCR)鉴别方法,为复杂组分中动物源性成分的鉴定提供示范。方法:根据猪源性鉴别引物,建立PCR鉴别方法,优化反应体系,并对此方法进行考察和验证。利用建立的PCR鉴别方法,对20批自制猪胆粉药材,19批市售猪胆粉药材和22批含猪胆粉中成药的猪源性成分进行鉴别,将市售的猪胆粉药材和含猪胆粉中成药PCR扩增的阳性产物进行酶切验证和序列测定验证。结果:20批自制猪胆粉药材、猪胆粉对照药材均能扩出约212 bp的特异性鉴别条带,牛和羊参考品均无条带; 19批市售猪胆粉药材中仅5批扩出特异性鉴别条带; 22批含猪胆粉中成药中有10批检出猪源性成分;猪胆粉对照药材及市售猪胆粉药材PCR扩增的阳性产物经Mnl I酶切后均能产生约200 bp的条带;猪胆粉及其中成药中扩增产物序列与Gen Bank数据库中相似性最高的物种为Sus scrofa,一致性为99%,与猪的序列高度一致。结论:该文建立的特异性PCR鉴别方法可准确鉴别猪胆粉及其中成药中的猪源性成分。

Specific PCR Method for Identification of Suis Fellis Pulvis and Its Chinese Patent Medicines

Objective: A polymerase Chain reaction(PCR) identification method for Suis Fellis Pulvis and its Chinese patent medicines was established to provide an example for the identification of animal-derived components in complex components. Method: A PCR identification method was established based on swine derivatives identification primers,the reaction system was optimized,and the established method was investigated and verified. By the established PCR identification method,the swine derivatives of 20 batches of self-made Suis Fellis Pulvis material,19 batches of commercially available Suis Fellis Pulvis and 22 batches of Chinese patent medicines containing Suis Fellis Pulvis were identified. The commercially available Suis Fellis Pulvis material and Chinese patent medicines containing Suis Fellis Pulvis positive products that were amplified PCR were verified by enzyme digestion and sequencing. Result: Totally 20 batches of self-made Suis Fellis Pulvis material and Suis Fellis Pulvis control material could expand the specific identification band of about 212 bp,and there was no bands in bovine and ovine reference, only 5 batches of the 19 batches of commercially available Suis Fellis Pulvis had expanded specific identification bands, 10 batches of 22 batches of Chinese patent medicines containing Suis Fellis Pulvis were detected to have swine derivatives, the Suis Fellis Pulvis control material and the PCR-amplified commercially available Suis Fellis Pulvis material positive products can produce about 200 bp of bands after digestion with Mnl I. The highest similarity between the amplification products sequence of Suis Fellis Pulvis and its Chinese patent medicines, and the GenBank database was Sus scrofa,the consistency was 99%,which conformed to the sequence of swine. Conclusion: The PCR identification method established in this paper can accurately identify the biological origin of Suis Fellis Pulvis and its Chinese patent medicines.