Rapid and sensitive identification of buffalo’s, cattle’s and sheep’s milk using species-specific PCR and PCR–RFLP techniques

Abstract

For the rapid, specific and sensitive identification of buffalo’s, cattle’s and sheep’s milk, species-specific PCR and PCR–RFLP techniques were developed. DNA from small amount of fresh milk (100 μL) was extracted to amplify the gene encoding species-specific repeat (SSR) region and the mitochondrial DNA segment (cytochrome- b gene). PCR amplification size of the gene encoding SSR region was 603 bp in both buffalo’s and cattle’s milk, while in sheep’s milk was 374 bp. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) technique was used to discriminate between buffalo’s and cattle’s milk. Restriction analysis of PCR–RFLP of the mitochondrial cytochrome- b segment (359 bp) analysis showed difference between buffalo’s and cattle’s milk. Where, the fragment length (bp) generated by TaqI PCR–RFLP were 191 and 168, whereas no fragments were obtained in cattle’s milk for cytochrome- b gene (359 bp). The proposed PCR and PCR–RFLP assays rep resent a rapid and sensitive method applicable to the detection and authentication of milk species-specific.