Harnessing Multiplex Polymerase Chain Reaction Assay for Convenient and Simultaneous Differentiation of Testudinis Carapax et Plastrum from Trionycis Carapax

Objective: The objective of the study was to develop a multiplex polymerase chain reaction (PCR) approach to simultaneously differentiate five Chelonia species and identify adulteration of two natural products. Materials and Methods: Five novel species‑specific primers were designed for CR, TS, MS, PS, and AF, followed by optimization of PCR conditions and validation for specificity and sensitivity. Then, a deliberate mixture was analyzed to verify the capability of adulteration detection. Finally, it was used to examine commercial products. Results: The developed method proved to be highly specific and detection limit was 1 ng for all the species tested. Particularly, it was still applicable and reliable when processed products or deliberate adulteration were examined. Two batches of commercial raw TCP products were identified to be counterfeited by TS using the newly proposed approach. Conclusion: The newly proposed multiplex PCR method showed sufficient merits to be readily employed as a regular means to authenticate edible and medicinal TCM products made of TCP and TC.