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

Authors:	Sachinandan De Biswajit Brahma Shamik Polley Ayan Mukherjee Deepak Banerjee Moloya Gohaina Karan Pratap Singh Rameswar Singh Tirtha Kumar Datta Surender Lal Goswami

Affiliation:	1. Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana 132001, India;2. KVK, SKUAST-Jammu, Bhaderwah, Jammu 182221, India;3. Division of Dairy Microbiology, National Dairy Research Institute, Karnal, Haryana 132001, India;1. Dipartimento di Agraria, Università degli Studi di Napoli Federico II, via Università 100, Parco Gussone, I-80055 Portici, Italy;2. Proteomics & Mass Spectrometry Laboratory, ISPAAM, National Research Council, via Argine 1085, I-80147 Naples, Italy;2. Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, United Kingdom;3. School of Biological, Biomedical and Environmental Sciences, University of Hull, Hull HU6 7RX, United Kingdom;1. Animal Genomics Lab., Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, Haryana, India;2. Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan;3. ICAR-Central Institute for Research on Goats, Mathura, Uttar Pradesh, India;4. The Royal Veterinary College, University of London, Hawkshead Lane, North Mymms, Hatfield, AL9 7TA, UK;1. Interfibio, Nanobiotechnology & Bioanalysis Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007 Tarragona, Spain;2. Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Strasse 96a, 07743 Jena, Germany;3. Chemical and Veterinary Investigation Office Stuttgart, Schaflandstrasse 3/2, 70736 Fellbach, Germany;4. Department of Chemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203, 21589 Jeddah, Saudi Arabia;5. Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys 23, 08010 Barcelona, Spain

Abstract:	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.

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