Polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial cytb gene from species of dairy interest.
Species identification plays an important role in food allergy prevention and food substitution detection that can reduce the commercial value of a product. For these reasons, many molecular methods have been developed to determine species origin; among them, polymerase chain reaction (PCR)-based methods were successfully applied to processed or unprocessed foodstuffs. An updated PCR-RFLP (restriction fragment length polymorphism) method of the cytb gene was developed for the identification of the 4 species of main interest in the dairy industry (Bos, Ovis, Capra, Bubalus). The comparative analysis of the 92 cytb sequences available in the database belonging to the 4 species allowed identification of 2 highly conserved regions, which were used to design 2 oligonucleotides for the PCR amplification of a 275 base-pair (bp) cytb fragment. The in silico analysis allowed identification of a set of species-specific restriction endonucleases (HaeIII, TaqI, and MwoI), which generated easily analyzable species-specific restriction profiles of the 275 bp cytb DNA fragment. The system was developed for both purified DNA and DNA extracted from meat or dairy products and finally tested on mixed samples, indicating its applicability to foodstuffs.
Lanzilao
Irene
I
Dipartimento di Biologia Animale e Genetica, Via Romana 19, 50125 Florence, Italy.
Burgalassi
Francesca
F
Fancelli
Silvia
S
Settimelli
Mario
M
Fani
Renato
R
D000818:Animals; D001483:Base Sequence; D002020:Buffaloes; D002417:Cattle; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D019295:Computational Biology; D003573:Cytochrome b Group / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D003612:Dairying; D030541:Databases, Genetic; D004587:Electrophoresis, Agar Gel; D006041:Goats; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012689:Sequence Homology, Nucleic Acid; D012756:Sheep; D012984:Software; D013696:Temperature
D003573:Cytochrome b Group; D004247:DNA; D004262:DNA Restriction Enzymes