Fluorescence Visual Identification of Four Main Varieties of Tibetan Medicinal Berberis cortex Based on Site-Specific PCR Technology.

Background: Berberis cortex is a typical multi-origin species in Tibetan medicine, with varying medicinal effects among different species. Establishing a rapid and accurate identification method for the major species of Tibetan medicinal B. cortex, including Berberis vernae Schneid. (SY), Berberis diaphana Maxim. (XH), Berberis kansuensis Schneid. (GS), and Berberis dictyophylla Franch. (CHZ), is conducive to the quality control of B. cortex.

Methods: Site-specific PCR visualization using SYBR Green I was developed based on rbcL SNP sites for SY, GS, and CHZ. For XH, which cannot be differentiated through rbcL and other barcoding SNP sites, RAPD-PCR methodology was employed to screen for species-specific sequences. A site-specific amplification visualization system was subsequently developed based on the identified sequence.

Results: The developed site-specific PCR visualization systems demonstrated excellent sensitivity, with all systems capable of detecting genomic DNA at concentrations as low as 100 fg. These systems were employed to analyze 16 batches of actual B. cortex samples. The analysis revealed that four samples were identified as SY, six samples as GS, two samples as CHZ, and three samples as XH. All results were concordant with sequencing data. One remaining negative sample was confirmed through sequencing as adulterated with Phellodendri Chinensis Cortex. Further authentication of 10 batches of Tibetan patent medicines containing B. cortex revealed that 2 batches contained both SY and GS, one batch contained both SY and CHZ, three batches contained exclusively GS, and three batches contained XH.

Conclusion: A site-specific PCR fluorescence visual identification system has been developed for the authentication of four major B. cortex species, enabling accurate identification of botanical origins in both B. cortex crude materials and Berberis-containing Tibetan patent medicines.