Single-Laboratory Validation of a Two-Tiered DNA Barcoding Method for Raw Botanical Identification

Abstract

Background: The applications of deoxyribonucleic acid (DNA) barcoding methods have been extended from authenticating taxonomic provenance of animal products to identifying botanicals used as herbal medicine and in botanical dietary supplements. DNA barcoding methods for botanical identification must be adequately validated to meet regulatory compliance. Objective: The goal of this study is to provide a validation protocol for a two-tiered DNA barcoding method that aims to identify raw botanicals. Methods: A barcode database was computationally validated to define the barcode combinations that can unambiguously identify botanicals in the database. A maximum variation sampling technique was used to capture a wide range of perspectives relating to DNA barcode-based botanical identification, including plant parts and species distance, for the experimental validation. Twenty-two authenticated botanicals were purposively sampled from different plant parts—covering both closely related and distantly related species—to validate the two-tiered DNA barcoding method. The performance of the method was assessed on accuracy, precision, ruggedness, and uncertainty. Results: High accuracy (100%) and precision (1.0) were obtained from the validation samples. The method was also found to be rugged and have acceptable uncertainty. Conclusions: The method was validated and suitable for DNA-based identification of botanical raw materials listed in the current database. Highlights: This work will provide support guidance for manufacturers and regulatory policy makers to implement equivalent validated and compliant DNA-based testing in quality control processes to improve botanical raw material identification and authentication.

Field Identification of <em>Matricaria chamomilla</em> using a Portable qPCR System

“…In addition, to align field testing results with other analytical techniques, field conclusions were further confirmed by a previously validated DNA barcoding method 25 (data not shown).…”

Development and Validation of a Species-specific PCR Method for the Identification of Ginseng Species Using Orthogonal Approaches

“…Ginseng root samples in commerce were authenticated by a previously validated DNA barcoding method 18 . Briefly, rbcL and ITS2 sequences of the root samples were amplified by a pair of primers with M13 tails (rbcL forward 5′- ATG TCA CCA CAA ACA GAA AC-3′, rbcL reverse 5′- GTA AAA TCA AGK CCA CCR-3′; ITS2 forward 5′- ATC CGA TAC TTG GTG TGA AT-3′, ITS2 reverse 5′- GAC GCT TCT CCA GAC TAC AAT-3′ synthesized by IDT) and bidirectionally sequenced using GeneAnalyzer 3500 from Applied Biosystems.…”

“…Among them, species-specific PCR methods are rising as an alternative to nontargeted universal DNA barcoding. In contrast to DNA barcoding that involves amplification, sequencing, and matching DNA barcodes against curated databases 18 , 19 , species-specific PCR, designed based on the characteristic genomic sequence of a species, has shown the potential to identify species efficiently without sequencing 20 , 21 . This easy-to-use feature allows species-specific PCR to be integrated into the current quality control system with minimal cost.…”