Validation of a Real-Time PCR Assay for Identification of Fresh and Processed 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. 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 C 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.