A rapid and accurate discrimination of Hedyotis diffusa Willd. from its adulterants

This study aimed to establish the molecular biology analytical methods based on the ladder-shaped melting temperature isothermal amplification (LMTIA) and the proofreading enzyme-mediated probe cleavage (Proofman), and two dual-LMTIA methods were developed for detection of H. diffusa and H. corymbosa as well as H. diffusa and H. pinifolia, the specific LMTIA primers and probes targeting the Internal Transcribed Spacer (ITS) sequences of H. diffusa, H. corymbosa, and H. pinifolia were designed. The Proofman-LMTIA technique was validated for specificity, optimized for temperature, determined for sensitivity and repeatability, and tested with commercial samples. The results showed that the established duplex Proofman-LMTIA method for H. diffusa vs. H. corymbosa could be finished in 30 min at the optimal temperature of 58°C with high specificity, the sensitivity of 10 pg/μL genomic DNAs H. corymbosa and the detection limit of 1%. For the other duplex system targeting H. diffusa vs. H. pinifolia, the amplification was finished within 20 min at the optimal temperature of 69°C with high specificity, the sensitivity was 50 pg/μL gDNA of H. pinifolia, and the detection limit was 1%. For the applicability test of the established duplex Proofman-LMTIA method, 10 commercial dried whole herb samples labeled as H. diffusa were analyzed. Among them, 3 samples tested positive for H. diffusa, 6 contained both H. diffusa and H. corymbosa, 1 contained H. corymbosa only, and H. pinifolia was not detected. Summarily, H. diffusa, H. corymbosa and H. pinifolia could be rapidly and accurately detected by the established Proofman-LMTIA methods.