Next-generation sequencing and targeted quantitative real-time polymerase chain reaction for detection of in the traditional Chinese medical formula Longdan Xiegan Wan.

Background: (Mu Tong) is commonly misused by (Guan Mutong) and (Chuan Mutong), which are nephrotoxic and carcinogenic. However, in the Pharmacopoeia of the People's Republic of China (2015 Edition), the method for determining remains undefined.

Methods: We used DNA barcode-based next-generation sequencing (NGS) combined with quantitative real-time polymerase chain reaction (qPCR) to detect in Longdan Xiegan Wan (LDXGW) for the first time. Compared with chromatographic studies, NGS enables better evaluation of the ingredient components of traditional Chinese medicine (TCM) preparations. The feasibility of qPCR using species-specific primers to determine the authenticity of species has been validated. In this study, the constituents of in LDXGW from three different manufacturers were scanned by NGS. The independently developed qPCR detection primers of , , and were specifically used to analyze the LDXGW mentioned above.

Results: The results showed that qPCR detected in all commercial samples. Meanwhile, NGS detected the counterfeit species (Tie-Xian Lian) in all samples. We found that qPCR shows a difference in detecting , but it was not able to identify the unknown additives and adulterants for the primer pairs of .

Conclusions: Hence, it is sensitive and rapid, qPCR is not suitable for detection alone. The NGS approaches offer important novel insights that complement the qPCR method. The combination of NGS and qPCR will be a powerful complement to traditional identification methods of TCM substances.