Variations in herb dosage due to species adulteration and dosing inaccuracies can substantially affect clinical safety and efficacy. Accurate species quantification remains challenging, as current methods often yield inconsistent results. This study introduces a novel pyrosequencing-based technique, termed herb molecular quantification (Herb-Q), designed to precisely quantify herbal products. We evaluated its effectiveness using Pinellia ternata and five of its adulterants. Initially, we assessed commonly used DNA barcodes with sequences from a public database, identifying two candidate regions, Maturase K (matK) and internal transcribed spacer 2 (ITS2), for screening specific single nucleotide polymorphism (SNP) loci, allowing for species-specific identification. These loci were validated by amplifying and sequencing genomic material from collected samples. Our validation studies showed that Herb-Q demonstrated excellent linearity, accuracy, repeatability, and detection limits. We established quantitative standard curves with high R2 values (> 0.99) to enable precise species quantification, which were combined with external standards to provide clear and accurate visual quantification results. The average bias in quantifying the tuber of P. ternata was 2.38%, confirming that Herb-Q can accurately identify and quantify herbal product constituents. Moreover, the entire quantification process took less than 4 h. This study presents a novel, rapid method for accurately quantifying species in herbal products and advances the application of DNA barcoding from species identification to quantitative detection.
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Funding
Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences(CI2021A04106)
Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences(CI2021A03910)
National Key Research and Development Program of China(2019YFC1710601)
Fundamental Research Funds for the Central Public Welfare Research Institutes of China(ZZ15-YQ-033)
Fundamental Research Funds for the Central Public Welfare Research Institutes of China(ZXKT21026)
Fundamental Research Funds for the Central Public Welfare Research Institutes of China(ZXKT23004)
Major Special Project of Scientific and Technological Cooperation of Bijie(2021-02)
Advantageous Chinese Medicinal Materials R&D Talent Base Project of Bijie, Guizhou Province(RCJD2020-21)
Bijie Technology Innovation Platform and Talent Team Bikehe[2023](66-BJZDSYS 2024-05)
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