Equivalence of droplet digital PCR and quantitative PCR in the molecular quantification of Shougong for the quality control of Jinlong capsule

Molecular quantification has recently been proposed as a new strategy with notable potential for quality control of animal-derived crude drugs in Chinese Patent Medicine. In this study, molecular quantitative assays for detecting Shougong (Gekko swinhonis) components as a quality control method for Jinlong Capsule (JLC) via droplet digital PCR (ddPCR) and real-time quantitative PCR (qPCR) were developed, achieving both qualitative and quantitative purposes. The primers and probe were designed targeting at cytochrome C oxidase subunit I (COI) gene. The established ddPCR and qPCR assays were validated through specificity, sensitivity, repeatability, stability assessments and inspection of simulated JLC preparation. Finally, Shougong component in commercial JLC samples were detected using the two assays. The primers and probe designed could successfully distinguish G. swinhonis from its adulterant. The two assays exhibited a good linearity in the sensitivity assay and were validated methodologically, suggesting the developed methods were specific, effective and reliable. Both assays were successfully applied in Shougong-specific amplicon determination in 10 batches of JLC samples. The results measured by ddPCR and qPCR showed a high degree of correlation (Spearman r = 0.9171, p < 0.05) and an excellent agreement assessed by Bland-Altman analysis. Overall, the ddPCR is substantially equivalent to the qPCR in the molecular quantitative determination of Shougong in JLC, and both of them can be applied to JLC quality control.