Dry reagent-based multiplex real-time PCR assays for specific identification of chicken, mutton, beef and pork in raw and processed meat products

DNA-based methods are more reliable than the conventional protein-based techniques and have been applied to species identification in food of animal origin and meat fraud detection. We developed and evaluated dry reagent-based real-time PCR assays for identification of four commonly eaten meat species. A set of species-specific primers and probes were designed targeting transforming growth factor beta-3 (TGFB3), prolactin receptor (PRLR), NADH dehydrogenase (ND5), and beta actin (ACTB) genes to differentiate chicken, mutton, beef and pork, respectively. All the real-time PCR reagents along with the primers and probes were lyophilized into a pellet form in the presence of suitable stabilizers. The assay specificity was tested with the DNA of animal species other than the target species and also with plant species. The assay was found to be sensitive down to 0.002 ng of DNA from each target species. Thermostability studies of lyophilized mix showed that it was stable for 90 days at room temperature and 120 days at 4 degrees C. When the method was applied for the analysis of commercial meat samples (n = 66), the results showed that seven samples contained non-declared meat components. The developed assay has the potential to monitor routine meat species identification from raw and cooked meat products and the method can also be implemented in food-testing laboratories.

Automated summary

DNA-based real-time PCR assays were developed for identification of four commonly consumed meat species with high sensitivity and stability, showing potential for routine monitoring of meat species in commercial meat products.