Meat species identification by real time PCR
Kumari, Rajni (2007) Meat species identification by real time PCR. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat - 388 001.
Full text available as:
A reliable and sensitive method for identification and differentiation of buffalo meat from mixed meats, particularly containing cattle meat is not currently available and is highly warranted. Present study was carried out to develop a Real Time PCR based test for identification and differentiation particularly of cattle and buffalo meat. DNA extraction from meat samples (ten each ) from cattle , buffalo, sheep, goat and chicken procured from slaughter house /market or obtained through biopsy were utilized for molecular study . Mitochondrial cyt b gene was amplified by conventional PCR using primers reported by Rea et al. (2001). PCR was optimized with respect to annealing temperature and primer concentration to give species specific amplification. A common forward and cattle specific reverse primer amplified 113 bp fragment on cattle DNA while common forward with buffalo specific reverse primer amplified 152 bp fragment. PCR did not produce any cross specific amplification. Further, it did not produce any amplification from other meat species studied. When the primers were used in duplex PCR, it amplified only the target species DNA i.e. either cattle or buffalo, while it amplified both the species DNA on cattle buffalo mixed meats. The optimized PCR was converted to the Real Time PCR using SYBR green Dye. In Real Time PCR the common forward primer with cattle specific reverse primer showed melting peak at 76.2 0C on cattle DNA while the common forward primer with buffalo specific reverse primers showed melting peak at 78.2 0C on buffalo DNA. Even in duplex PCR it showed only species specific melting peaks in respective species DNA. But when duplex PCR was evaluated on cattle- buffalo mixed DNA template in equal proportion it exhibited two peaks, a major buffalo specific and a minor cattle specific, merging into one broader peak at 78.2 0C coinciding with buffalo specific melting point. However it was possible to know presence of mixed DNA by Real Time PCR using duplex primers. The duplex Real Time PCR showed only a single broader peak at 78.2 0C at 1: 10 and all further ratios. Hence an independent cattle specific Real Time PCR was run on mixed DNA which produced cattle specific melting peak at 76.2 0C upto 1: 1000 ratios. At 1: 10,000 ratio it did not showed any cattle specific melting peak. Thus, it was possible to detect and differentiate cattle meat mixed in buffalo meat upto 1: 1000 fraction i.e. 9 pg of cattle DNA adulterated in buffalo DNA by running a duplex PCR followed by cattle specific Real Time PCR. Duplex Real Time PCR did not produce any amplification and melting peaks on DNA templates from sheep, goat and chicken. Thus, Real Time PCR was found to be successful in differentiating cattle and buffalo mixed meat samples. Consequently, Real Time PCR assay developed in the present study was found to be very sensitive and specific to detect adulteration of cattle meat in buffalo meat such that it can be adopted in an advanced lab like forensic lab.
Archive Staff Only: edit this record