Harnessing multiplex polymerase chain reaction assay for convenient and simultaneous differentiation of testudinis carapax et plastrum from trionycis carapax

Articles

Abstract
Pharmacognosy Magazine,2020,16,69,393-399.
Published:June 2020
Type:Original Article
Authors:
Author(s) affiliations:

Zhaoqun Jiao1, Liqun Chen1, Lijuan Xia1, Yaya Yang1, Yang Zheng1, Hongxia Chen2, Liwei Wang1, Pingtian Yu1, Yuping Shen1, Huan Yang1
1 Department of Chinese Materia Medica and Pharmacy, School of Pharmacy, Jiangsu University, Zhenjiang, Jiangsu, China
2 Department of Plant Biology, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, USA

Abstract:

Background: Testudinis Carapax et Plastrum (TCP, Guijia) and Trionycis Carapax (TC, Biejia), which made from the shell of Chinemys reevesii (CR) and Pelodiscus sinensis (PS), were two kinds of widely used animal-derived Traditional Chinese medicine (TCM). Some cheap substitutes such as the carapace and plastron of Trachemys scripta (TS), Mauremys sinensis (MS), or Apalone ferox (AF) shells obtained from restaurants are sometimes used, which expose the public health to a high risk and cause unfair competitions in the market. 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.

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Images
 Three percent agarose gel showing the analytical sensitivity of  multiplex polymerase chain reaction
Keywords