Development of Multiplex PCR Based Method for detection of Species Specific Milk

Abstract

The adulteration of food products is a significant problem in the food processing and production. Most frequently, such products are adulterated that are produced in big quantities and further, the expensive products whose adulteration brings a profit. This is how fraudulent producers try to cheat consumers and authorities. Adulteration of costly milk with cheaper one becomes a matter of concern for research workers. Cow milk is frequently used for adulteration because of its prevailing production in the world and its lower price as compared to other types. The present study was carried out for detection of milk species with the use of cytochrome b and 12S r RNA gene variability by multiplex PCR. Milk samples from Cow, Buffalo, sheep and goat were utilized for molecular analysis. The milks of these principal dairying species are well characterized. Genomic DNA was isolated from four samples of each species. Mitochondrial cytochrome b and 12S r RNA gene was amplified by conventional and multiplex PCR using a common forward primer and species-specific reverse primer. PCR cycling protocol included initial denaturation at 94 oc for 5 minutes then followed by 40 cycles of 94 oc for 30 seconds, 60°C for 30 seconds 72°C for 30 seconds and final extension at 72°C for 1 0 minutes. PCR amplicons were resolved by Agarose gel electrophoresis and for each species produce a characteristic band pattern in conventional and multiplex PCR. The PCR products showed species-specific DNA fragments of 500bp, 220bp, 155bp, 330 bp from Cow, Buffalo ,goat, and sheep respectively and will help traders and consumers against milk adulteration.