title: Development and validation of fast duplex real-time PCR assays based on SYBER Green florescence for detection of bovine and poultry origins in feedstuffs.
abstract: SYBR duplex real-time polymerase chain reaction (SDRT-PCR) with melting curve analysis was developed that can unite the reward of multiplex PCR with real-time PCR to recognize animal genes rapidly in feedstuffs. The method merges the use of bovine (Bos taurus) and poultry (Gallus gallus) specific primers that amplify small fragments (amplicons <200 base pairs) of the mitochondrial gene. Appropriate mixtures of bovine and poultry heat treated meat DNAs were used to develop the assay. Gene products of bovine and poultry were produced two distinct melting peaks simultaneously at 79.5 °C and 87.5 °C, respectively. Multiplex analysis of the reference feed samples showed that the detection limit of the assay was 0.001% for bovine and poultry species. Based upon the assay results it has been concluded that SDRT-PCR assay might be an efficient tool for the verification of species origin in feedstuffs.
authors.affiliation: Department of Medical Biology and Genetics, University of Gaziantep, Gaziantep, Turkey. Electronic address: drmuhammadsafdar8@gmail.com.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Safdar
authors.affiliation: Department of Medical Biology and Genetics, University of Gaziantep, Gaziantep, Turkey; Department of Chemistry, Fatih University, Buyukcekmece, 34500 Istanbul, Turkey.
authors.forename: Yasmeen
authors.initials: Y
authors.lastname: Junejo
mesh_terms: D000821:Animal Feed / Q000032:analysis*; D000818:Animals; D052160:Benzothiazoles; D002417:Cattle / Q000235:genetics; D004272:DNA, Mitochondrial / Q000032:analysis*; D003959:Diamines; D008460:Meat / Q000032:analysis*; D009930:Organic Chemicals; D011200:Poultry / Q000235:genetics; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D052160:Benzothiazoles; D004272:DNA, Mitochondrial; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I
keywords: Duplex analysis; Feedstuffs; Real-time PCR; SYBR Green dye