猪胆粉及其中成药的特异性PCR鉴别方法
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摘要
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。
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.
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.
引文
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[9]黄璐琦,唐仕欢,李军德,等.动物药材分子鉴定研究策略[J].中国中药杂志,2011,36(3):234-236.
[10]陈蓉,吴成丽,邓赟,等.DNA分子鉴定技术在中成药真伪性鉴别中的应用与研究进展[J].中药与临床,2016,7(2):83-86.
[11]崔占虎,龙平,王颖莉,等.DNA分子标记技术在中成药鉴定中的应用与展望[J].中药材,2015,38(1):188-192.
[12]苟惠,王译伟,郑茜,等.含当归中成药的DNA提取及其分子鉴定[J].中国实验方剂学杂志,2018,24(1):44-50.
[2]石岩,耿颖,郑天骄,等.猪胆粉HPLC-ELSD全轮廓谱图主要胆汁酸测定及化学计量学分析[J].药物分析杂志,2017,37(2):283-289.
[3]黄璐琦,袁媛,袁庆军,等.中药分子鉴定发展中的若干问题探讨[J].中国中药杂志,2014,39(19):3663-3667.
[4]杨晶凡,蒋超,袁媛,等.快速PCR方法在山药真伪鉴别中的应用[J].中国实验方剂学杂志,2018,24(22):45-49.
[5]吴桂凡,张文娟,程显隆,等.特异性PCR方法鉴别蛇胆汁及其伪品[J].中国中药杂志,2018,43(10):2053-2056.
[6]Kwon K R,Baek S I,Choi S H.Identification of Fel ursi and Cattle and Pig Bile Juicesby species-specific PCRand PCR-RFLP[J].J Pharmacopunct,2009,12(1):13-20.
[7]蒋超,黄璐琦,袁媛,等.《中国药典》动物药材基原物种中文名和拉丁学名引证规范[J].中国科学:生命科学,2018,48(7):772-782.
[8]石岩,魏锋,马双成.猪胆药用研究进展及质量控制概况[J].中国中药杂志,2018,43(4):637-644.
[9]黄璐琦,唐仕欢,李军德,等.动物药材分子鉴定研究策略[J].中国中药杂志,2011,36(3):234-236.
[10]陈蓉,吴成丽,邓赟,等.DNA分子鉴定技术在中成药真伪性鉴别中的应用与研究进展[J].中药与临床,2016,7(2):83-86.
[11]崔占虎,龙平,王颖莉,等.DNA分子标记技术在中成药鉴定中的应用与展望[J].中药材,2015,38(1):188-192.
[12]苟惠,王译伟,郑茜,等.含当归中成药的DNA提取及其分子鉴定[J].中国实验方剂学杂志,2018,24(1):44-50.