Validation of a Targeted PCR Method for Raw and Processed Botanical Material Identification: An Example Using Matricaria chamomilla (Chamomile)

Abstract

Background: A requirement of current good manufacturing practices for dietary supplements is that manufacturers must identify their dietary ingredients. DNA-based methods can provide species-level authentication that may sometimes be difficult to achieve using conventional morphological and chemical analysis methods. However, because of varying levels of DNA degradation in botanical materials, many commercial tests fail to generate consistent test results across all types of botanical materials. AOAC published guidelines for validation of botanical identification methods and proposed probability of identification (POI) as a method performance parameter. However, few DNA-based botanical authentication methods in the literature follow these guidelines and evaluate POI. Objective: To provide a targeted PCR method validation example that follows AOAC guidelines for validation of botanical identification methods. Methods: Using Matricaria chamomilla (chamomile) as an example, we performed a single-laboratory validation for a targeted PCR method that aimed to identify both raw and processed chamomile materials. The performance parameters of the test were evaluated by carrying out an inclusivity/exclusivity study and a Specified Superior Test Material/Specified Inferior Test Material study to demonstrate that the method’s POI meets industry requirements. Results: The chamomile samples were identified by the method and achieved a POI greater than 0.9 with respect to all types of chamomile botanical materials. Conclusions: The method was validated for DNA-based identification of raw and processed chamomile materials, such as sterilized powders and extracts. Highlights: This work will provide insight for laboratories and manufacturers that aim to develop and validate DNA-based botanical identification methods.

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

“…Efforts have been made to target DNA fragments according to their degradation level. Studies have shown that species-specific DNA can be amplified from certain processed botanical materials, such as Matricaria chamomilla and Lonicera japonica extracts, and even can be used to differentiate between different varieties of P. ginseng in certain extracts 21 , 29 , 30 , 31 .…”

“…In addition, the application of traditional DNA barcoding should be limited to unprocessed or minimally processed plant materials since DNA degradation in processed materials could result in false-negative results when using universal primers to target barcodes typically longer than 400 bp 22 , 23 . Species-specific PCR, when designed and validated to amplify short DNA fragments (usually less than 200 bp), may have advantages for potentially addressing species identification in heavily processed botanical materials containing partially degraded DNA, such as extracts 21 , 22 . With increased coverage on processed botanical materials, species-specific PCR could be applied to more types of botanical materials in commerce, addressing species identity issues in powders and extracts, which were once difficult to be achieved by other methods.…”

“…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.…”

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

“…should also not show a false positive due to the specificity of the assay, according to a previous validation study40 .Copyright © 2020 JoVE Creative Commons Attribution-NonCommercial-NoDerivs 3.…”