Genetic Identification of Botanical Species in Complex Herbal Products via High-throughput DNA Barcoding

Introduction: Dietary supplements are facing increased scrutiny from government regulators due to systemic issues with contamination, adulteration, and outright fraud. These supplements often have dozens of ingredients making label verification difficult. Chemical profile analysis depends on the age and part of the plant being tested. Traditional Sanger sequencing can identify only one gene from one species at a time making the identification of mixed products unfeasible. These limitations can be overcome by using longer read shotgun sequencing technology to process multi-target amplicon pools.

Purpose: To develop an efficient test strategy for identifying botanical ingredients from plant species that are prevalent in commercial dietary supplements, herbal remedies, spices, and other health products. 

Methods: Pharmaceutically and economically important botanical species (342) were collected and 5 barcoding genes from these plants were analyzed by high-throughput shotgun sequencing.  The resulting reads were compared with public data from NCBI to determine the optimal target genes for identification. Species-specific PCR assays were developed to identify 150 plant species that are most frequently encountered in the herbal product market. Individual plant samples, mixed plant samples and commercial products were screened and the results confirmed by species-specific PCR analysis. 

Results: An in-house database of ~320,000 sequences with valid species names was extracted from NCBI. All 342 botanical species sequenced were taxonomically classified using this dataset. Our analysis revealed that overall 71% of them were correctly identified to the genus or species level, and 98% were identified to the family level.  Potential adulterants were detected in multi-organism and commercial samples. Species-specific PCR effectively confirmed the genetic analysis and real-time PCR provided quantitative information when needed. 

Significance: It is feasible to correctly identify botanical species by sequencing pools of DNA barcodes. Limited sensitivity of identification due to inadequate coverage of species in public databases was resolved by sequencing the missing species in-house. The genetic identification of complex botanical samples will become a potent tool for verifying ingredient labels and screening for potential contaminants and adulterants in commercial products.