Rapid and Simple Identification of Pork in Meat Products by Using the Isothermal Target and Probe Amplification Assay

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. For example, pork DNA was found in halal chicken sausages served in a primary school in Westminster, central London, the local authority had stated on 14 March 2015. PCR is one of the most widely used techniques in diagnostic applications because it allows a sensitive and rapid diagnosis. However, this technique is not suitable for routine identification of pork in meat products because of the need for expensive thermal cycler equipment and complex operations. The isothermal target and probe amplification (iTPA) analysis has been developed for identifying pork species in meat samples. In this study, the isothermal target and probe amplification assay was developed for rapid, simple and highly specific identification of pork DNA. For the assay, the mitochondrial 16S ribosomal RNA gene and the FRET-based signal probe were amplified at 61oC, followed by measurement of the fluorescent signal. As little as 1 pg of template DNA could be detected without any cross-reactivity with non-target species. Meat mixtures prepared by mixing pork meat with other meats at different ratios (0.01% 10%) were tested, and the iTPA assay allowed detection of as little as 0.01% pork in meat mixtures. Thus, this work showed that the iTPA assay could be used for specific identification of pork species. This assay only requires a heat block and a fluorescence reader for amplification and detection, respectively, and it has great potential for use as a hand-held device or point-of-care-testing system. The iTPA assay is sensitive and specific for rapid screening of fraudulent adulteration/substitution of meat products.