Rapid Identification of Gentiana rigescens Franch. Using Proofman Probe Ladder-Shape Melting Temperature Isothermal Amplification (Proofman-LMTIA) Technology.

Introduction: Gentiana rigescens Franch. (Jianlongdan) and other Gentiana species (Longdan), including G. manshurica, G. scabra, and G. triflora, are valuable medicinal plants used in traditional Chinese medicine. However, their similar appearances and potential for market misidentification necessitate a rapid and accurate identification method.

Objective: This study aimed to develop a rapid identification method using Proofman-LMTIA technology to accurately identify Jianlongdan and prevent misidentification in the market.

Methods: Specific primers and Proofman probes were designed based on the differential region of Internal Transcribed Spacer 2 (ITS2) sequence between Jianlongdan and Longdan. The method's specificity, sensitivity, detection limit, and stability were evaluated, and its applicability to commercial gentian products was tested.

Results: The developed Proofman-LMTIA method demonstrated high specificity and sensitivity for Jianlongdan at an optimal reaction temperature of 56°C, with a detection limit of 10 pg/μL and the ability to identify 0.1% Jianlongdan in mixtures. The method showed good stability and was successfully applied to market samples.

Conclusion: The Proofman ladder-shape melting temperature isothermal amplification (Proofman-LMTIA) method offers a rapid, specific, and cost-effective tool for the authentication of Jianlongdan, crucial for ensuring the quality of herbal medicine and protecting consumers from misidentification and potential adulteration.