| Abstract
| - Real-time uniplex and duplex polymerase chain reaction (PCR) assays with a SYBR Green I post-PCR melting curve analysis were evaluated for the identification and quantification of bovine, porcine,horse, and wallaroo DNA in food products. Quantitative values were derived from threshold-cycle(Ct) data obtained from serial dilutions of purified DNA. The limits of detection in uniplex reactionswere 0.04 pg for porcine and wallaroo DNA and 0.4 pg for cattle and horse DNA. Species specificityof the PCR products was tested by the identification of peaks in DNA melting curves, measured asthe decrease of SYBR Green I fluorescence at the dissociation temperature. The peaks could bedistinguished above the background even at the lowest amount of template DNA detected by the Ctmethod. The system was also tested in duplex reactions, by use of either single-species DNA orDNA admixtures containing different shares of two species. The minimum proportions of each DNAspecies allowing the resolution of Tm peaks in the duplex reactions were 5% (cattle or wallaroo) incattle/wallaroo mixtures, 5% porcine and 1% horse in porcine/horse mixtures, 60% porcine and 1%wallaroo in porcine/wallaroo mixtures, and 1% cattle and 5% horse in cattle/horse mixtures. A lossin the sensitivity of the method was observed for some DNA combinations in the duplex assay. Incontrast, the results obtained from SYBR Green I uniplex and duplex reactions with single-speciesDNA were largely comparable to those obtained previously with species-specific TaqMan probes,showing the suitability of that simpler experimental approach for large-scale analytical applications. Keywords: Food authenticity; species identification; real-time PCR; cytochrome b; meat species
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