Development and Application of qPCR Assay for Species-specific Identification of Two Animal-derived Tonics: Vinegar-quenched Testudinis Carapax et Plastrum and Vinegar-quenched Trionycis Carapax

Abstract

Vinegar-quenched Testudinis Carapax et Plastrum (TCP) and vinegar-quenched Trionycis Carapax (TC) are made from the shells of Chinemys reevesii and Trionyx sinensis. However, some cheap substitutes from non-medicinal species such as Trachemys scripta, Mauremys sinensis, Chelydra serpentina, and Apalone ferox are often seen on the market, which exposes public health to high risk. Conventional methods are not applicable for the identification of these two products since their intrinsic constituents are largely destroyed after being highly processed. A new quantitative polymerase chain reaction (qPCR) method was developed to detect DNA components of Vinegar-quenched TCP and vinegar-quenched TC based on simple sequence repeat differential genes of animal-derived mitochondria. Six species-specific primer reactions were designed and further validated for specificity, sensitivity, and repeatability. Finally, the developed method was used to assess the authenticity of their commercial products. The assay exhibited good specificity, and the limit of detection was above 1 × 102 copies/µL. There was good linearity in the concentration, ranging from 1 × 102 to 1 × 107 copies/µL. In addition, the method was validated through repeatability assessment (coefficient variation <1%). While qPCR was applied for the analysis of 36 batches of commercial products of these two tonics, it could effectively identify the authenticity of the products, and consequently, seven batches were fake or adulterated. This newly proposed method is promising for the quality evaluation of highly processed animal-derived Chinese medicines, which will assist in ensuring safety and efficacy in clinical practice and protect fair trade in the market.