Abstract：
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.
摘要：
由于物种掺假和剂量不准确导致的草药剂量变化会严重影响临床安全性和有效性。准确的物种量化仍然具有挑战性，因为目前的方法往往会产生不一致的结果。这项研究介绍了一种基于焦磷酸测序的新技术，称为草药分子定量(Herb-Q)，旨在精确量化草药产品。我们使用半夏及其五种掺假品评估了其有效性。最初，我们用公共数据库中的序列评估了常用的DNA条形码，确定两个候选区域，成熟酶K（matK）和内部转录间隔区2（ITS2），用于筛选特定的单核苷酸多态性（SNP）基因座，允许特定物种的识别。通过对来自收集的样品的基因组材料进行扩增和测序来验证这些基因座。我们的验证研究表明，Herb-Q表现出良好的线性，准确度,重复性,和检测限。我们建立了高R2值（>0.99）的定量标准曲线，以实现精确的物种定量，与外部标准相结合，以提供清晰准确的视觉定量结果。定量假单胞菌块茎的平均偏差为2.38%，确认Herb-Q可以准确地识别和量化草药产品成分。此外，整个量化过程花费不到4小时。这项研究提出了一种新颖的，准确定量中药产品中物种的快速方法，推进了DNA条形码从物种鉴定到定量检测的应用。
