TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures

A rapid and highly specific real-time quantitative PCR, based on the amplification of a fragment of the mitochondrial 12S ribosomal RNA gene (rRNA), has been developed for the quantitation of pork (Sus scrofa) in binary pork/beef muscle mixtures. The method combines the use of pork-specific primers, that amplify a 411 bp fragment from pork DNA, and mammalian-specific primers amplifying a 425-428 bp fragment from mammalian species DNA, which are used as endogenous control. An internal fluorogenic probe (TaqMan), that hybridizes in the pork-specific and also in the mammalian DNA fragments is used to monitor the amplification of the target gene. A comparison of the cycle number (C-t) at which mammalian and pork-specific PCR products are first detected, in combination with the use of reference standards of known pork content, allows the determination of the percentage of pork in a mixed sample. Analysis of experimental pork/beef muscle binary mixtures demonstrated the specificity and sensitivity of the assay for detection and quantitation of pork in the range 0.5-5%. (c) 2005 Elsevier Ltd. All rights reserved.