Rapid identification of Radix Achyranthis Bidentatae and Radix Cyathulae ingredients using Proofman-LMTIA method

Abstract

Radix Achyranthis Bidentatae (RAB) and Radix Cyathulae (RC) share the same name “Niu-Xi”, which are two types of well-known herbal medicines in Asian countries due to their long history and extensive pharmacological activities. However, the misuse of counterfeits and confused species of RAB or RC due to the increased demands might lead to improper efficacies and serious health risks to consumers. To address the challenge, this study devises a rapid and accurate method named Proofman-LMTIA for the identification of RAB and RC. A set of species-specific primers were selected to target internal transcription spacer (ITS) gene sequences of RAB and RC. The temperatures of the Proofman-LMTIA systems were optimized, and the specificities, sensitivities and detection limits were investigated. The detection of RAB and RC ingredients based on the Proofman-LMTIA method was finished in less than 20 min. The sensitivities of RAB and RC DNA templates were 50 pg/µL and 10 pg/µL, respectively. The detection limits of RAB and RC DNA samples extracted from artificially adulterated formula granules were 0.1 %. To evaluate the applicability and effectiveness of the Proofman-LMTIA systems, 16 formula granules were collected and analyzed for RAB or RC authentication, among these, 14 out of 16 samples tested positive for RAB or RC components. Overall, the Proofman-LMTIA technique could be a reliable and suitable method for the accurate and rapid identification of RAB and RC ingredients in commercial samples. The popularization of the LMTIA technique will protect the rights of consumers and maintain the stability of the medicine market.