Simplex and duplex PCR assays for species specific identification of cattle and buffalo milk and cheese

A polymerase chain reaction, amplifying a fragment of the mitochondrial DNA D loop region was developed for species specific detection of cattle and buffalo milk. The method was simultaneously extended for detection of HTST pasteurized milk samples and cheeses of bovine and buffalo origin. A common forward primer was used with two different species specific reverse primers that resulted amplification of a 126 bp and 226 bp products for cattle and buffalo, respectively, in simplex as well as in multiplex polymerase chain reaction. The primers successfully amplified DNA extracted by conventional protocol from minimal amount of raw milk, heat treated milk and cheese of either bovine or buffalo origin. The primers showed a high degree of specificity. The sensitivity of the assay was excellent with detection level of 0.1 percent adulteration of cow and buffalo milk or cheese (0.15 ng buffalo and 0.04 ng cattle DNA). The assay represents a sensitive and simple method for identification of adulteration in milk and cheese.