Identification and quantitation of species in complex dna mixtures by real-time polymerase chain reaction.

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Lopez-Andreo M, Lugo L, Garrido-Pertierra A, Prieto MI, Puyet A.

Departamento de Bioquimica y Biologia Molecular IV, Facultad de Veterinaria, Universidad Complutensede Madrid, 28040 Madrid, Spain.

"Six TaqMan real-time polymerase chain reaction (PCR) systems using minor groove binding (MGB) probes have been developed for thedetection quantitation of bovine, porcine, lamb, chicken, turkey, and ostrich DNA in complex samples. Species-specific amplification was achieved by combining only twofluorogenic probes and 10 oligonucleotide primers targeting mitochondrial sequences, decreasing the cost of the assay significantly. The limits of detection ranged from 0.03to 0.80 pg of template DNA. Analysis of experimental mixtures containing two to four different species showed the suitability of the assay for detection of more than 1%of pork, chicken, or turkey and of more than 5% of cattle or lamb. The quantitation accuracy in samples containing 10-100% of beef or pork DNA was close to 90%. Thesystem is complemented with one additional TaqMan MGB detector based on consensus sequence segments of the nuclear 18S ribosomal RNA gene. A method to evaluate the presenceof unknown eukaryotic DNA in a mixture, where data derived from the species-specific detection are compared with the experimental values obtained from the general 18Sdetector, is presented. This method allows the validation of the quantitative measurements, providing an internal control of the total content of PCR-amplifiable DNA inthe sample. The system was tested on DNA mixtures containing different shares of up to four different species and on DNA extracted from processed commercial food samples."
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