ABSTRACT
The European Community ban on use of meat and bone meal in ruminant feed, as a consequence of the spread of bovine spongiform encephalopathy in Europe, has prompted a number of investigations about the possibility of detecting animal tissues in feedstuff. In this paper, a study on vertebrate primers, designed in the 16S rRNA gene of mitochondrial DNA, is described. These primers were able to amplify fragments that contained between 234 and 265 bp. The fragments were specific for bovine, porcine, goat, sheep, horse, rabbit, chicken, trout, and European pilchard and were confirmed by sequence analysis amplicons. The primers were used in a PCR assay applied to five samples of meat and blood meals of different species and subjected to severe rendering treatments (134.4 to 141.9 degrees C and 3.03 to 4.03 bar for 24 min). The presence of vertebrate tissues was detected in all samples. The assay proved to be rapid and sensitive (detection limit 0.0625%). It can be used as a routine method to detect animal-derived ingredients in animal feedstuff.
Subject(s)
Animal Feed/analysis , DNA/analysis , Encephalopathy, Bovine Spongiform/prevention & control , Meat Products/analysis , Polymerase Chain Reaction/methods , Animals , Cattle , Chickens , DNA Primers , DNA, Mitochondrial/analysis , Encephalopathy, Bovine Spongiform/transmission , Fishes , Gene Amplification , Goats , Horses , Humans , Molecular Weight , RNA, Ribosomal, 16S/analysis , RNA, Ribosomal, 16S/genetics , Rabbits , Reproducibility of Results , Sensitivity and Specificity , Sequence Alignment , Sequence Homology, Nucleic Acid , Sheep , SwineSubject(s)
Animal Feed/analysis , DNA Primers , Polymerase Chain Reaction/methods , Animals , Cattle , Chickens , Databases, Nucleic Acid , Encephalopathy, Bovine Spongiform/prevention & control , Encephalopathy, Bovine Spongiform/transmission , Fishes , Food Microbiology , Horses , Insecta , RNA, Ribosomal, 16S/genetics , Rabbits , SwineABSTRACT
A duplex polymerase chain reaction (PCR) was developed to identify the milk of bovine and buffalo species in cheese products, particularly in mozzarella cheese, a typical Italian cheese made from buffalo's milk. Two sets of primers were designed on the basis of the alignment of the sequence codifying mitochondrial cyt b available in the GenBank database. The primers proved to be species-specific, giving rise to 279-bp (bovine) and 192-bp (buffalo) amplified fragments. Since the amplification conditions for bovine and buffalo primers were identical, a duplex PCR was successfully applied to identify the two species in a single reaction step. This technique, when used to test cheese products from the retail trade, allowed the detection of partial or even total substitution of cow's milk for buffalo's milk, in some cases in samples of cheese misleadingly labeled "pure buffalo" mozzarella.
Subject(s)
Cheese/analysis , Animals , Base Sequence , Buffaloes , Cattle , Food Labeling , Gene Amplification , Milk/chemistry , Molecular Sequence Data , Polymerase Chain Reaction/methods , Sequence Alignment , Species SpecificityABSTRACT
A new type of hemoglobin F, in which isoleucine in position 75 (E 19) of the gamma chain is replaced by a threonine residue, has been found in 29 out of 32 homozygotes for beta thalassemia. The amount of this hemoglobin ranges from traces to 40% of the total Hb F. The same gamma75 Thr chain is also present in the Hb F of 40% of normal newborns and premature infants examined, of one 14-week-old fetus and in one out of 3 patients with aplastic anemia and raised levels of Hb F. Our results strongly suggest that the synthesis of this new chain is under the control of a gamma gene nonallelic with those coding for Agamma and Ggamma chains.