Use of cytochrome b gene variability in detecting meat species by multiplex PCR assay
Jain, Shally (2005) Use of cytochrome b gene variability in detecting meat species by multiplex PCR assay. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, INDIA.
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Food authenticity is currently an issue of major concern for food authorities, since incorrect labeling of animal foods may have remarkable negative consequences. To circumvent this problem, molecular methods had been developed. The present study was carried out for detection of meat species with the use of cytochrome b gene variability by multiplex PCR. Meat samples from cattle, buffalo, sheep, goat, chicken, pig and horse were utilized for molecular analysis. Genomic DNA was isolated from six samples of each species as per method described by Ausubel et al. (1987) with some modifications. Mitochondrial cyotchrome b gene was amplified by conventional and multiplex PCR using a common forward primer and species-specific reverse primer. Multiplex PCR was carried by mixing of primers in the different ratio viz. 1: 0.2: 3: 3: 0.6: 3: 0.6: 2 for forward: Chicken: Goat: Cattle: Cattle: Sheep: Pig: and horse specific reverse primers. PCR cycling protocol included initial denaturation at 95° C for 5 minutes then followed by 34 cycles of 95 ° C for 30 seconds, 60 °C for 30 seconds 72 °C for 30 seconds and final extension at 72° for minutes. PCR amplicons were resolved by agarose gel electrophoresis and for each species produce a characteristic band pattern in conventional and multiplex PCR was obtained. The PCR products showed species-specific DNA fragments of 157, 227, 274, 274, 331, 398 and 439 bps from goat, chicken, cattle, buffalo, sheep, pig and horse respectively. Species-specific DNA fragments were amplified from the tissue samples cooked at 100º C and 120º C except for horse meat cooked at 120º C. The cattle and buffalo fragment was weakly amplified from DNA extracted from cooked meat at 120º C sample. Putrefied meat samples showed successful amplification of mitochondrial cytochrome b gene using species specific primers by multiplex PCR. Putrefication did not inhibit detection potential of the test and same results were obtained as in fresh meat samples. Semi – quantification of pork and beef DNA mixed in different ratio could be done by this method. The intensity of amplified product band increased as theconcentration of DNA template increased. The PCR efficiencies calculated was 1.00 with correlation 0.82 and 0.99, respectively for cattle and pig. The multiplex PCR could detect upto 0.09 ng of DNA of meat species. Thus, the multiplex PCR was found to be a simple reliable and sensitive and highly specific test for simultaneous detection of meat species.
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