The Asian Journal of Animal Science

Mayura R. Patil ¹, Anilkumar S. Bannalikar ¹, Swati R. Gunjal ², Preeti H. Admane ²

Affiliations
1 Department of Animal Biotechnology, Bombay Veterinary College, Mumbai (M.S.), IN
2 Department of Biotechnology, University of Mumbai, Vidyanagri Campus, Kalina, Mumbai (M.S.), IN

Abstract

Meat is one of the major sources of proteins for the human beings. Adulteration of good quality meat with poor is one of the widespread problems of the meat industry. The PCR based methods developed in the recent times are suggested to facilitate precise and quick detection of meat animal species overcoming the disadvantages of conventional methods. The present investigation was undertaken with a view to evaluate the efficacy of PCR-RFLP analysis of mt 12S rRNA gene in identification of major meat animal species viz., cattle, buffalo, sheep and goat. Amplification of 456 bp of mt. 12S rRNA gene by PCR from meat samples of all the species followed by restriction digestion of the PCR products with AluI, HhaI, BspTI and ApoI in separate reactions. Characteristic RFLP patterns were generated specific for each species that allowed their accurate identification. The method was also evaluated for its efficacy in identification of species from adulterated meat samples. Buffalo meat: sheep meat (mutton) and buffalo meat: goat meat (chevon) samples were mixed in different proportions (viz., 50:50, 60:40, 70:30, 80:20, 90:10 and 95:05) in a manner similar to that of unadulterated samples. Among the adulterated meat samples, accurate identification of both the species was possible in combinations: 50:50, 60:40, 70:30 and 80:20 consistently in 15 experiments. The results of present study indicated that the PCR-RFLP analysis of mt 12s rRNA gene could be potentially useful tool for detection of meat animal species.

Keywords

Meat Species, Adulteration, mt 12S rRNA Gene, PCR-RFLP, Restriction Enzyme.

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Mayura R. Patil ¹, Anilkumar S. Bannalikar ¹, Vikas D. Dighe ²

Affiliations
1 Department of Animal Biotechnology, Bombay Veterinary College, Mumbai (M.S.), IN
2 National Institute for Research in Reproductive Health, Parel, Mumbai (M.S.), IN

Abstract

Brucellosis is a zoonotic disease caused by the bacteria of the genus Brucella that produce infections leading to abortion and infertility, and recurrent fevers in humans. The disease is endemic in many areas of the world. Thus, we designed a TaqMan-based 5'nuclease-real-time PCR for molecular diagnosis by targeting the 702bp deleted sequence of eryC gene from B. abortus S19 that allowed the specific quantitative detection of Brucella wild strains but not the vaccine strain B. abortus S19. The ery C gene which encodes the enzyme d-erythrulose-1-phosphate dehydrogenase that plays an important role in the ery thritol metabolism. This carbohydrate promotes the growth of some strains and is present in the placenta. The assay proved to be 100 per cent specific, as determined with Brucella isolates and reference Brucella strains, and highly sensitive with excellent linearity and PCR efficiency. When implemented on blood samples, the real time PCR assay detected higher proportion (80%) of positive samples than conventional bcsp31 PCR (70%) and i-ELISA (65%).

Keywords

Brucella spp., Vaccine Strain B. abortus S19, ery C Locus, 5' Nuclease PCR.

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