A Poultry Universal Primer-Based Fluorescent PCR (PUP-fPCR) for Simultaneous Identification and Quantification of...

To combat poultry meat adulteration, we developed a poultry universal primer-based fluorescent PCR (PUP-fPCR). Through comprehensive genomic alignment analysis, a poultry-specific nuclear DNA sequence containing phylogenetically conserved regions and hypervariable segments with interspecies nucleotide polymorphisms was employed to develop universal primers targeting conserved flanking sequences and TaqMan probes for hypervariable segments. Then, a multiplex quantitative PCR method incorporating universal primers with four TaqMan probes was developed with high specificity and sensitivity (limit of detection: 0.005 ng). Analytical performance evaluation using prepared DNA mixtures revealed robust accuracy (relative deviation: 0.80-5.05%) and precision (relative standard deviation: 0.94-13.84%). This single-tube multiplex system leverages the spectral discrimination of TaqMan probes to simultaneously detect four poultry species, overcoming primer competition issues inherent in conventional multiplex PCR designs. This integrated approach reduces system complexity while maintaining detection efficiency, providing regulatory agencies with a robust tool for combating meat adulteration and ensuring food quality supervision.