STANDARDIZATION AND SPECIES IDENTIFICATION OF PRESSURE COOKED MEAT BY POLYMERASE CHAIN REACTION
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Date
2017
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College of Veterinary and animal Science,Mannuthy
Abstract
Meat authenticity has economical, religious and public health concerns. Hindus and Muslims are strictly against the consumption of beef and pork on religious grounds, respectively. To punish under Food Safety & Standards Act, 2006 for mislabelling it is very much necessary to prove under court of law, for which DNA based technique for meat species identification is highly authentic.
In the present study PCR based techniques based on forensically Informative Nucleotide Sequencing (FINS) and PCR-RFLP for authentication of fresh and pressure cooked meat of cattle (Bos indicus X Bos taurus), buffalo (Bubalus bubalis), sheep (Ovis aries), goat (Capra hircus), pig (Sus scrofa) and rabbit (Oryctolagus cuniculus) and fat samples of above said species except rabbit have been standardized. Six meat samples from each above said species collected in duplicate along with four fat samples also collected from above said species except rabbit. One set of six each species meat samples were pressure cooked at 105ÂșC (15lbs for 15 min.).
DNA was extracted from fresh, pressure cooked meat and fat samples by following standard protocols. A set of universal primers VPH-F and VPH-R were selected for targeting mitochondrial cytochrome b (mt cyt-b) gene (168-777 bp). PCR amplification yielded amplicon of 609 bp in all type of samples from all species studied. Amplicons obtained from pressure cooked meat samples were sequenced and aligned using Basic Local Alignment Search Tool (BLAST) of National Centre for Biotechnological Information (NCBI). Targeted cyt-b sequence obtained from pressure cooked beef, buffalo beef, chevon, mutton, pork and rabbit meat showed 99, 100, 100, 100, 99 and 99 per cent similarity respectively with in the species. The partial cyt-b gene sequences obtained from above mentioned samples were submitted to gene bank with accession no MF503652, MF503653, MF503654, MF503655, MF503656 and MF574101 respectively. FINS successfully authenticate pressure cooked beef, buffalo beef, chevon, mutton, pork
and rabbit meat and differentiated same from each other and indicate which species the meat belongs too.
PCR-RFLP technique is economical one for routine meat identification. So, PCR amplicons obtained from fresh, pressure cooked meat as well as fat samples were restricted digested with TaqI and AluI restriction enzymes resulted in a pattern that could authenticate and differentiate beef, buffalo beef, chevon, mutton, pork and rabbit. TaqI was found to yield single fragment in case of rabbit (609 bp), two fragments in cattle (294 and 315 bp), buffalo (70 and 539 bp), sheep (294 and 315 bp) and pig (266 and 299 bp) and three fragments in goat (131, 163 and 272 bp). Cattle and sheep was further differentiated by AluI restriction enzyme it was found to yield two fragments in buffalo (92 and 517 bp), sheep (259 and 350 bp) and pig (261 and 321 bp), three fragments in case of cattle (64, 92 and 453 bp) and single fragment (609 bp) in case of rabbit and goat. RFLP pattern of restriction enzymes used in this study was highly specific and specificity did not vary within the species and processing temperatures of the meat and fat samples.
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