Qi Wentao, Tuerxun Julaiti, Li Jianchao, Wang Chen, Luo Rong, Liu Changli
1School of Traditional Chinese Medicine, Capital Medical University, 10 You'anmen Outer West 1st Street, Beijing, 100069 China.
Beijing Key Laboratory of Traditional Chinese Medicine Collateral Disease Theory Research, Beijing, 100069 China.
3 Biotech. 2020 Jun;10(6):240. doi: 10.1007/s13205-020-02233-1. Epub 2020 May 8.
The traditional Chinese medicine Bupleuri radix (chaihu) is the dried roots of and and many adulterants exist because of the differences in traditional understanding, medication habits and seed resources. Therefore, rapid and accurate identification methods for (Apiaceae) seeds are required. We analyzed the internal transcribed spacer (ITS) sequences of five common species to detect variations in them, including , , var, and var. Based on single nucleotide polymorphisms (SNPs) in the ITS region, we designed five specific primer pairs and used these primers in an allele-specific PCR technique to establish a robust molecular identification method. The neighbor-joining (NJ) tree of ITS sequences showed that five species formed their own monophyly. Five specific primer pairs were designed and integrated into a specific PCR master mix and cycling conditions. The primer pair of BCF/R8 for could amplify a specific identification band of 429 bp and the minimum detection limit of the 5 g mixture was about 5%; BSF/R11 for could amplify a specific 464 bp band and the minimum detection limit was about 5%; BMSF/R1 for var could amplify a specific 344 bp band and the minimum detection limit was about 1%; BFF/R7 for could amplify a specific 137 bp band and the minimum detection limit was about 1%; BSPF/R1 for var. could amplify a specific 390 bp band. Subsequent analysis proved the reliable accuracy and good practicability of the five specific identification primers, indicating that the allele-specific primer PCR identification method can quickly identify seeds. The method elaborated in this study has the advantages of simple operation, good accuracy and high efficiency.
中药柴胡是柴胡属植物的干燥根，由于传统认识、用药习惯和种子资源的差异，存在许多伪品。因此，需要快速准确的柴胡属（伞形科）种子鉴定方法。我们分析了五种常见柴胡属物种的内部转录间隔区（ITS）序列，以检测它们之间的变异，包括柴胡、狭叶柴胡、北柴胡变种、锥叶柴胡和膜缘柴胡变种。基于ITS区域的单核苷酸多态性（SNP），我们设计了五对特异性引物，并将这些引物用于等位基因特异性PCR技术，以建立一种可靠的分子鉴定方法。ITS序列的邻接（NJ）树显示，五个柴胡属物种形成了各自的单系。设计了五对特异性引物，并将其整合到特异性PCR预混液和循环条件中。柴胡的BCF/R8引物对可扩增出一条429 bp的特异性鉴定条带，5 g混合物的最低检测限约为5%；狭叶柴胡的BSF/R11引物对可扩增出一条464 bp的特异性条带，最低检测限约为5%；北柴胡变种的BMSF/R1引物对可扩增出一条344 bp的特异性条带，最低检测限约为1%；锥叶柴胡的BFF/R7引物对可扩增出一条137 bp的特异性条带，最低检测限约为1%；膜缘柴胡变种的BSPF/R1引物对可扩增出一条390 bp的特异性条带。后续分析证明了这五对特异性鉴定引物的可靠准确性和良好实用性，表明等位基因特异性引物PCR鉴定方法可以快速鉴定柴胡属种子。本研究阐述的方法具有操作简单、准确性好、效率高的优点。
