A high-throughout PCR test strip method for the rapid identification of four placentas

Deer placenta and Placenta Hominis are valued traditional Chinese medicines often adulterated with cheaper pig or cow placenta. Current identification methods frequently lack the throughput, speed, or simplicity for effective field monitoring. This study aimed to develop a high-throughput, rapid method to simultaneously identify deer, human, pig, and cow placenta. The approach involved screening a rapid genomic DNA extraction protocol and designing specific primers for the mitochondrial cytochrome b gene of each species, with primers labeled with FAM and Biotin. Following the optimization of PCR amplification conditions, results were visually detected using immunocolloidal gold test strips. The method was rigorously evaluated for its specificity, reproducibility, sensitivity, and stability. The results showed that the extracted DNA was of satisfactory concentration, purity, and integrity. Under optimized conditions (59 °C annealing temperature, 20 cycles), authentic samples for all four placenta types produced two distinct red bands on the test strips, while adulterated and blank controls showed only one control band. Agarose gel electrophoresis confirmed specific amplification for each target with no cross-reactivity. The method demonstrated strong specificity, excellent reproducibility and stability, and a high sensitivity of 0.01 ng μL^-1, surpassing conventional electrophoresis. In conclusion, this PCR-based test strip method enables the visual, simultaneous authentication of the four placenta types in a single test, making it highly suitable for on-site rapid monitoring and quality control.