REAL-TIME PCR ASSAY FOR HIGHLY SPECIFIC DETERMINATION OF PORK IN RAW AND HEAT TREATED MEAT MIXTURES

摘要

IntroductionNumerous analytical methods have been developed for species identification of animal tissues in meat products to protect the consumer from the illegal and undesirable adulteration for economical, religious and health reasons. The methods based on the detection of species-specific proteins such as electrophoresis, isoelectric focusing (IEF), enzyme-linked immunosorbent assays (ELISA) are proved to be inadequate (less sensitive) and often not suitable for the species identification of meat products which had been previously exposed to very high temperatures causing denaturation of the proteins (Meyer et al., 1994). However, methods of DNA analysis based on the polymerase chain reaction (PCR) offer a potential for the doubtless detection of the animal species used, even for the products that have been subject to intensive processing with complex composition (Meyer et al., 1993). Conventional PCR techniques allow the qualitative detection of different animal species in a mixture, but they are not appropriate to achieve the quantification of the species tissue in a product (Rodriguez et al., 2005). Real time PCR is widely accepted as a robust assay for the species identification and quantification of nucleic acid molecules due to its high sensitivity and specifity, large dynamic range of detection and a low carry-over contamination risk (Mackay et al., 2002). In this technique, amplification of the target gene is monitored by an increased fluorescence signal which enables direct assesment of the results after the PCR application without additional detection steps. In this paper, a TagMan real-time PCR assay was studied to optimize the determination of pork in heat treated meat mixtures, added fraudulently to the products and consequently verify the concordance of the labels.