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

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.