A quick and simple method for the identification of meat species and meat products by PCR assay

Identification of meat species by TaqMan-based real-time PCR assay

Genetic traceability of livestock products: A review

Molecular biology techniques have been used for species identification in food of animal origin in relatively recent years. A polymerase chain reaction (PCR) based method, the multiplex PCR, was recently applied to species identification in meat and meat products. It allows co-amplification of separate regions of a single gene or specific fragments, each typical of a different animal species in a single PCR reaction, using different pairs of primers in the same reaction mix. In the present paper, the duplex-PCR technique is proposed to identify bovine and water buffalo DNA in a single PCR assay in milk and mozzarella cheese (a typical Italian cheese, originally made from pure water buffalo milk). Because of its lower cost, undeclared bovine milk is added to water buffalo milk for making different kinds of mozzarella cheese. The results of this experiment indicate the applicability of this method, which showed an absolute specificity for the two species and a high sensitivity even down to low DNA concentrations (1 pg). In bovine and water buffalo mixtures of both milk and mozzarella cheese, the minimum concentration tested was 1% of bovine in water buffalo milk and water buffalo in bovine milk. The importance of the somatic cell content in raw milk is also discussed with special reference to the evaluation of mixtures (milk or cheese) of the two species.

Use of duplex polymerase chain reaction (duplex-PCR) technique to identify bovine and water buffalo milk used in making mozzarella cheese

Alpha-connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the beta-connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on beta-connectin. Purified beta-connectin was digested by trypsin into 1700- and 400-kDa fragments. which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 x 10(7) M(-1) and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 microM. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of beta-connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of beta-connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl2 and 70 microM leupeptin to split connectin filaments into beta-connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200+/-33 kDa (mean+/-SD), while alpha- and beta-connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 microm at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N2 line region of parent filaments in situ. Because the secondary structure of the 400-kDa fragment was changed by the binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction-relaxation cycle of skeletal muscle.

Calcium binding to an elastic portion of connectin/titin filaments.

Polymerase Chain Reaction (PCR) was applied to species identification of meats and meat products. For the identification of meat species by PCR, three sets of primer, i.e., BF (5'-GGATCCCTCTTCTCGCTCCG-3') and BR (5'-GGTTAAGCTACATCAACGGG-3') for cattle, PF (BF) and PR (5'-ACTGAATAGCACCTTGTTTGG-3') for pig, and also CF (5'-CCCCTTTCCCCAGGGGGGGT-3') and CR (5'-AGGGGTTGCTGATCTCTCGT-3') for chicken, were designed according to the published mitochondrial D-loop DNA sequences from cattle, pig and chicken. Amplification was carried out 35 cycles of denaturation at 94 degrees C for 2 min, annealing at 66-68 degrees C for 2 min, and extension at 72 degrees C for 3 min in the presence of Taq DNA polymerase. Products amplified were analyzed by 2% agarose gel electrophoresis. Products amplified from beef, pork and chicken gave single specific DNA fragment of 518 bp, 321 bp and 400 bp, respectively. Using this method, several meat products were analyzed. Mixing of beef, pork or chicken in meat products was revealed by identification of each single specific DNa fragment. When three sets of primers were employed together, beef, pork and/or chicken specific DNA fragment from these mixed meat products could be identified by only one PCR reaction. Sheep is closely related species of cattle, but lamb does not gave and amplified DNA fragment under these experimental conditions. The detection limits in a mixed DNA sample were approximately 0.1% beef in pork, 0.001% pork in beef, 1% chicken in pork, respectively

Species identification of meats and meat products by PCR

The binding of paratropomyosin to β-connectin, which has been suggested to interact at the A-I junction of a sarcomere, was confirmed by measuring the changes in turbidity of a mixture with changing NaCl concentration, pH and free calcium ions, and by morphological observation and a coprecipitation assay of the aggregates formed in the mixture. Paratropomyosin also bound to the 400-kDa fragment which is the N-terminal portion of β-connectin and contains the A-I junction region. Moreover, the interaction of paratropomyosin with the 400-kDa fragment was enhanced by a calcium ion concentration from 10-7 M to 10-5 M and markedly suppressed above 10-4 M calcium ions. We conclude that paratropomyosin probably binds to the 400-kDa fragment of β-connectin in the A-I junction region in living and pre-rigor skeletal muscle. In postmortem skeletal muscle paratropomyosin may be released from the 400-kDa portion of the connectin filament by increased calcium ion concentration and translocated on to thin filaments to i...

Interaction of Paratropomyosin with β-Connectin and Its 400-kiloDalton Fragment from Chicken Skeletal Muscle as Influenced by the Calcium Ion Concentration

We found that paratropomyosin bound to β-connectin, in examining binding of paratropomyosin at the junction of A- and I-bands of sarcomeres. The turbidity of a mixture of β-connectin and paratropomyosin was greater with more paratropomyosin added, but high concentrations of Ca2+ suppress this increase. These results suggest that paratropomyosin is released from connectin filaments at the A-I junction region by increased concentrations of calcium ions in postmortem skeletal muscles.

https://www.tandfonline.com/doi/pdf/10.1271/bbb.62.821

Binding of Paratropomyosin to β-Connectin from Chicken Skeletal Muscle

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Species identification of meats and meat products by PCR

Interaction of Paratropomyosin with β-Connectin and Its 400-kiloDalton Fragment from Chicken Skeletal Muscle as Influenced by the Calcium Ion Concentration

Binding of Paratropomyosin to β-Connectin from Chicken Skeletal Muscle