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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. [ABSTRACT FROM AUTHOR]

Background: Asini Corii Colla (ACC; donkey-hide glue) and Taurus Corii Colla (TCC; bovine-hide glue) are popular healthcare food supplements as well as well-recognized medicine in Traditional Chinese medicine clinics. Due to large demand of them and limited resources of their animal origins, the skin of other animals are often used to make their fake or adulterated products. Objective: In this study, we aimed to develop a species-specific polymerase chain reaction (PCR) approach to detect deoxyribonucleic acid (DNA) fragments of ACC and TCC for rapid authentication purpose. Materials and Methods: Four sets of novel species-specific primer were designed, and PCR conditions were optimized for the PCR assay, which was further validated for specificity and sensitivity. Then, deliberate mixture was analyzed to further verify the capability of adulteration detection by the developed PCR method. Finally, it was used to assess the authenticity of commercial products. Results: Four primers' sets were specific for amplification of extracted mitochondrial DNA of donkey, bovine, swine, and horse when the annealing temperature was 59°C, 59°C, 63°C, and 65°C, and 40 thermal cycles was performed. Under this optimized PCR conditions, 1 ng/μL of horse DNA template has been detected in the sensitivity test, with 0.1 ng/μL for donkey, bovine, and swine. The assay was capable of detecting spiked species at 10% level in premixed ACC or TCC samples. Conclusion: The newly developed PCR-based technology was specific and sensitive, and it was a convenient approach for the authentication of commercial ACC and TCC products. [ABSTRACT FROM AUTHOR]

Background: Leech is a well-known animal-derived Chinese medicine; intentionally or unintentionally, interfusing fake products have exposed patients to high risk. Objective: In this study, we would like to find a rapid and accurate technology to identify medicinal leech. Materials and Methods: Three sets of species-specific primer were designed, and polymerase chain reaction (PCR) conditions were optimized for the four species analyzed. Specificity and sensitivity test were carried out, and the reference sample mixtures were analyzed. Finally, the developed method was used to assess the authenticity of commercially available products. Results: Three sets of species-specific primer were confirmed to have high interspecies specificity and good intraspecies stability. The limit of detection was estimated to be 1 ng for all of four assayed species. Subsequent validation demonstrated that the raw material, processed, and even highly processed products of leech can be conveniently authenticated with good sensitivity and precision by this newly established PCR-based method. Conclusion: These designed novel primer sets have shown distinct performance on the identification of adulterate products. The new technology needs no procedure for sequencing and has been successfully employed for the reliable authentication of raw material of leech and even their processed and highly processed products. [ABSTRACT FROM AUTHOR]