Detection of the Bovine Milk Adulterated in Camel, Horse, and Goat Milk Using Duplex PCR

Food ingredient adulteration, especially the adulteration of milk and dairy products, is one of the important issues of food safety. A rapid, simple, sensitive, specific, and low-cost DNA detection method was established to screen bovine milk adulterated in three different kinds of special milk (camel, horse, and goat milk). This method can detection both bovine milk and special milk in a single duplex polymerase chain reaction (duplex PCR). In this study, specific primers of duplex PCR were designed based on 16S-RNA genes from camel and bovine mitochondria and D-LOOP genes from horse and goat mitochondria. The designed primers had good specificity and could accurately amplify the target fragments (711 bp, 584 bp, 241 bp, and 184 bp). The duplex PCR was applied to the binary mixtures of raw milk fixed percentage and processed dairy products (freeze-dried, pasteurized, and ultra-high temperature (UHT) sterilized with the same mixtures and commercial samples). The limit of detection (LOD) of special milk adulterated with bovine milk was 0.1% in raw milk mixture. Pasteurized and UHT sterilized raised the LOD to 0.2% and 0.5%, but freeze-dried did not raise the LOD. Duplex PCR maintained preferable selectivity and sensitivity in the detection of samples with different processed, and it is expected to be used in market detection.