Droplet digital PCR-based approach for identifying Trionycis Carapax and its Chinese patent medicines.

Trionycis Carapax, the dorsal armour of Trionyx sinensis Wiegmann, is a traditional Chinese medicine (TCM) widely used in clinical applications. The substitution of high-value Trionyx sinensis Wiegmann with other soft-shelled turtles has caused market confusion and compromised the quality of proprietary Chinese medicines. To ensure drug safety and clinical efficacy, effective methods for identifying and quantifying adulterated Trionycis Carapax and its Chinese patent medicines are urgently needed. A droplet digital PCR (ddPCR) method targeting mitochondrial DNA for qualitative detection of Trionycis Carapax and its Chinese patent medicines. Processed and deeply processed products of Trionyx sinensis Wiegmann and its two common adulterants were analyzed for specificity, sensitivity, and utility using singleplex and quadruplex ddPCR assays. The ddPCR assay detected the target genes and identified low-abundance adulterants below 10 copies in unbalanced mixed samples, confirmed by Sanger sequencing. This work demonstrates the ability of ddPCR technology to detect TCM adulterants with complex composition and processing, providing a method for quality control of deep-processed animal-based TCMs with weak morphological characteristics.