Development of Multiplex PCR Assay for Species Identification of Cattle, Hog, Chicken and Duck from Raw Meats

Introduction: Species identification of animal tissues in meat products is an important issue to protect the consumer from illegal and/or undesirable adulteration; for economic, religious and health reasons. In this reason, accurate analytical methods are needed for the labeling of meat products with requiring simple and fast procedure

Purpose: In this study, a multiplex polymerase chain reaction (PCR) assay was developed for the simultaneous identification of four species of cattle, hog, chicken and duck from raw meats

Methods: The primers used in this study were designed in different regions of mitochondrial DNA (16S RNA) after alignment of the available sequences in the Genbank database. The primers generated specific fragments of 94 bp, 192 bp, 279 bp and 477 bp lengths for hog, chicken, cattle and duck, respectively. Thirty-six cycles of amplification were run using a Mastercycler system (Eppendorf) as follows: denaturation at 94°C for 30 sec, annealing at 60 °C for 30 sec, and extension at 72°C for 30 sec.

Results: The detection limit of the multiplex PCR assay was 1 pg of each template DNA extracted from raw meats of cow, pig, chicken and duck. Total 145 samples (cattle 55, hog 30, chicken 30, and duck 30) were tested for PCR assay. The species specificity was 100% in all four species in the multiplex PCR assay. When this PCR test was applied to other animal species of horse, sheep, goat and turkey, no amplification was shown.

Significance: These studies suggest that this multiplex PCR assay can be used for rapid and simultaneous species identification of cattle, hog, chicken and duck from raw meats. Further studies are needed for evaluation of developed multiplex PCR from various samples including processed meat products.