Validation of a Real-Time PCR Assay for Identification of Fresh and Processed Carica papaya Botanical Material: Using Synthetic DNA to Supplement Specificity Evaluation.

Several commercially important botanicals have a lack of diagnostic testing options that can quickly and unambiguously identify materials of different matrices. Real-time PCR can be a useful, orthogonal approach to identification for its exceptional specificity and sensitivity. Carica papaya L. is a species with a lack of available identification methods, and one which features two distinct commercially relevant matrices: fresh fruit and powdered fruit extract. In this study, we demonstrate the successful design and validation of a real-time PCR assay for detection of papaya DNA extracted from the two matrices. We also propose a technique that can be used during exclusivity panel construction, when genuine botanical samples are not available for certain species: substitution with synthetic DNA. We demonstrate the use of this material to complete a comprehensive specificity evaluation and confidently determine suitable Ct cutoff values. Further, we demonstrate how ddPCR can be used to determine the copy number of the target sequence in a set amount of genomic DNA, to which synthetic DNA samples can be corrected, and how it can verify specificity of the primers and probe. Through the presentation of successful assay validation for papaya detection, this work serves as a guideline for how to approach specificity evaluation when non-target botanical samples are difficult to obtain and otherwise may not have been included in the exclusivity panel.

Development of Hydrolysis Probe-Based qPCR Assays for Panax ginseng and Panax quinquefolius for Detection of Adulteration in Ginseng Herbal Products.

Authentication of Panax ginseng and Panax quinquefolius products is important to be able to mitigate instances of adulteration and substitution that exist within the international supply chain of ginseng. To address this issue, species-specific hydrolysis probe qPCR assays were developed and validated for both P. ginseng and P. quinquefolius herbal dietary supplements. Performance of the probe-based assays was evaluated using analytical validation criteria, which included evaluation of: (1) specificity, in selectively identifying the target species; (2) sensitivity, in detecting the lowest amount of the target material; and (3) repeatability and reproducibility of the method in detecting the target species in raw materials on a real-time PCR platform (reliability). The species-specific probes were developed and successfully passed the validation criteria with 100% specificity, 80-120% efficiency and 100% reliability. The methods developed in this study are fit for purpose, rapid, and easy to implement in quality assurance programs; authentication of ginseng herbal supplements is possible, even with extracts where DNA is fragmented and of low quality and quantity.

Validation of a Triplex Quantitative Polymerase Chain Reaction Assay for Detection and Quantification of Traditional Protein Sources, Pisum sativum L. and Glycine max (L.) Merr., in Protein Powder Mixtures.

Several botanicals have been traditionally used as protein sources, including the leguminous Pisum sativum L. and Glycine max (L.) Merr. While a rich history exists of cultivating these plants for their whole, protein-rich grain, modern use as powdered supplements present a new challenge in material authentication. The absence of clear morphological identifiers of an intact plant and the existence of long, complex supply chains behoove industry to create quick, reliable analytical tools to identify the botanical source of a protein product (many of which contain multiple sources). The utility of molecular tools for plant-based protein powder authentication is gaining traction, but few validated tools exist. Multiplex quantitative polymerase chain reaction (qPCR) can provide an economical means by which sources can be identified and relative proportions quantified. We followed established guidelines for the design, optimization, and validation of qPCR assay, and developed a triplex qPCR assay that can amplify and quantify pea and soy DNA targets, normalized by a calibrator. The assay was evaluated for analytical specificity, analytical sensitivity, efficiency, precision, dynamic range, repeatability, and reproducibility. We tested the quantitative ability of the assay using pea and soy DNA mixtures, finding exceptional quantitative linearity for both targets - 0.9983 (p < 0.0001) for soy and 0.9915 (p < 0.0001) for pea. Ratios based on mass of protein powder were also tested, resulting in non-linear patterns in data that suggested the requirement of further sample preparation optimization or algorithmic correction. Variation in fragment size within different lots of commercial protein powder samples was also analyzed, revealing low SD among lots. Ultimately, this study demonstrated the utility of qPCR in the context of protein powder mixtures and highlighted key considerations to take into account for commercial implementation.

Investigating appropriate molecular and chemical methods for ingredient identity testing of plant-based protein powder dietary supplements.

Plant-based protein powders are rapidly growing in popularity, and outdated quality assurance tools expose vulnerabilities to adulteration via different methods of "protein spiking". Adequate diagnostic tools are urgently needed to be able to authenticate protein source ingredients and screen for potential adulterants. We explored the application of three diagnostic tools for ingredient identification: targeted PCR with Sanger sequencing, NGS, and LC-MS/MS. We collected 33 samples of common commercial products from the plant-based protein powder market and sought to identify botanical components using the three technologies. We found success in detection with all approaches, with at least one main protein source being identified by at least one approach in all samples. The investigation uncovered challenges to data collection or result interpretation with each technology including but not limited to amplification biases with PCR technologies, potential influence of DNA degradation, and issues with protein solubility during isolation. Ultimately, each platform demonstrated utility along with certain caveats, which epitomized the importance of orthogonality of testing.

Exploring DNA quantity and quality from raw materials to botanical extracts.

OBJECTIVES: The aim of this study was to explore the variability in DNA quality and quantity along a gradient of industrial processing of botanical ingredients from raw materials to extracts. METHODS: A data matrix was assembled for 1242 botanical ingredient samples along a gradient of industrial processing commonly used in the Natural Health Product (NHP) industry. Multivariate statistics was used to explore dependant variables for quality and quantity. The success of attaining a positive DNA test result along a gradient of industrial processing was compared among four biotechnologies: DNA barcoding, NGS, Sanger sequencing and qPCR. RESULTS: There was considerable variance in DNA quality and quantity among the samples, which could be interpreted along a gradient from raw materials with greater quantities (50-120 ng/µL) of DNA and longer DNA (400-500bp) sequences to extracts, which were characterized by lower quantities (0.1-10.0 ng/µL) and short fragments (50-150bp). CONCLUSIONS: Targeted molecular diagnostic tests for species identity can be used in the NHP industry for raw and processed samples. Non-targeted tests or the use of NGS for any identity test needs considerable research and development and must be validated before it can be used in commercial operations as these methods are subject to considerable risk of false negative and positive results. Proper use of these tools can be used to ensure ingredient authenticity, and to avert adulteration, and contamination with plants that are a health concern. Lastly these tools can be used to prevent the exploitation of rare herbal species and the harvesting of native biodiversity for commercial purposes.

DNA Quality and Quantity Analysis of Camellia sinensis Through Processing from Fresh Leaves to a Green Tea Extract.

Background: Although there has been some success using DNA barcoding to authenticate raw natural health product (NHP) botanical ingredients, there are many gaps in our understanding of DNA degradation, which may explain low PCR and sequencing success in processed NHPs. Objective: In this study, we measured multiple DNA variables after each step in the processing of a green tea extract in order to document DNA quality and quantity. Methods: We sampled plant material after each step of green tea extract processing: five steps at a Chinese tea farm (n = 10) and five at an NHP processing facility (n = 3). We hypothesized that processing treatments degrade and remove DNA from NHPs, reflected by decreasing quantities of extractable genomic DNA (gDNA), an increasing proportion of small DNA fragments in genomic extracts, and decreasing quantitative PCR (QPCR) efficiency [higher cycle threshold (Ct) values]. DNA from end-production green tea extract was sequenced in order to try to validate material as the botanical of interest. Results: We saw a 41.1% decrease in mean extractable gDNA through farm processing (P < 0.01) and a 99.7% decrease through facility processing (P < 0.05). There was a 26.3% decrease in mean DNA fragment size through farm processing (P < 0.001) and an 82.0% decrease through facility processing (P < 0.05). QPCR efficiency was reduced through processing, marked by significant increases in Ct values with 100 base pair (bp) and 200 bp PCR targets (P < 0.05), and an inability to amplify 300 bp targets when using DNA template from end-production green tea extract. Conclusions: Although there was significant degradation and removal of DNA through processing, sufficiently intact DNA was able to be recovered from highly processed green tea extract for further sequencing and identification. Highlights: This work addresses a key gap in the understanding of DNA degradation through processing and provides useful information to consider when designing molecular diagnostic techniques for NHP identification.