Droplet digital PCR versus real-time PCR for in-house validation of porcine detection and quantification protocol: An artificial recombinant plasmid approach

Abstract

Authenticity and traceability are essential for modern food and medicine inspection, and reliable techniques are important for the trade of halal foods, which reach more than 20 percent of the world market. A sensitive and accurate porcine detection method is required to develop a conformity assessment system that includes laboratory testing for porcine-free certification. This study proposes a procedure that could be incorporated into the development of a standardized control and protocol for real-time PCR (qPCR) methods and their traceability using droplet digital PCR (ddPCR). The design used a recombinant pUC57 plasmid as an amplification target to carry the 97 bp fragment of the porcine ATCB gene. The absolute quantification and linearity assessment showed high precision with R2 values of 0.9971 and 0.9998 for qPCR and ddPCR, respectively. In general, both methods showed comparable results in terms of linearity and detection limit. However, both limit of detection assessments showed high sensitivity, although ddPCR showed a slightly higher sensitivity than that of qPCR, especially at low DNA concentrations. Multiple-sample and inter-participatory testing evaluations revealed a high sensitivity, broad applicability, and robustness of the qPCR method. Therefore, we conclude that based on a recombinant plasmid analysis with a low quantity (less than five copy number), the digital PCR method produced more reliable results. These results could provide scientific information for regulatory authorities, especially those in Indonesia, to consider the development and formulation of a well-established qPCR protocol for porcine detection using expected DNA concentrations.