Identification of Bupleurum (Apiaceae) seeds by allele-specific PCR based on ITS sequences

The traditional Chinese medicine Bupleuri radix (chaihu) is the dried roots of Bupleurum chinense and Bupleurum scorzonerifolium and many adulterants exist because of the differences in traditional understanding, medication habits and seed resources. Therefore, rapid and accurate identification methods for Bupleurum (Apiaceae) seeds are required. We analyzed the internal transcribed spacer (ITS) sequences of five common Bupleurum species to detect variations in them, including B. chinense, B. scorzonerifolium, B. marginatum var. stenophyllum, B. falcatum and B. smithii var.parvifolium. 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 Bupleurum 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 B. chinense 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 B. scorzonerifolium could amplify a specific 464 bp band and the minimum detection limit was about 5%; BMSF/R1 for B. marginatum var. stenophyllum could amplify a specific 344 bp band and the minimum detection limit was about 1%; BFF/R7 for B. falcatum could amplify a specific 137 bp band and the minimum detection limit was about 1%; BSPF/R1 for B. smithii var. parvifolium 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 Bupleurum seeds. The method elaborated in this study has the advantages of simple operation, good accuracy and high efficiency.