Species identification and quantification of silver pomfret using the droplet digital PCR assay

Abstract

Adulteration of the high-value silver pomfret (Pampus argenteus) is a serious problem worldwide, necessitating accurate identification and quantification of the species. In this study, optimisation of the digital droplet PCR (ddPCR) assay for the identification and quantification of the silver pomfret was carried out. The primer and probe concentrations, melting temperature, and PCR cycle number were optimised by combining single-factor experiments with an orthogonal experimental design. The absolute limits of detection and quantification of the ddPCR were 2copies/μl and 21 copies/μl, respectively. Its sensitivity was 0.1% for meat mixtures and 0.5% for DNA mixtures. The ddPCR was 156 times more sensitive than the real-time PCR, although both methods had similar specificities. However, the overall time needed to complete the ddPCR method was twice that of the real-time PCR. Notwithstanding, the ddPCR methodology established in this study can be a valuable tool for addressing species adulteration issues.