This assay was developed to have a cheaper, simpler and more rapid assay to detect meat species substitution. A novel direct lysis (DL) method was used to extract DNA directly from meat tissue, and to obtain a sample of DNA for multiplex PCR within 15 min, which is more rapid than using a commercial lysis kit. Four pairs of high-specificity primers for the mitochondrial D-loop region of beef, pork, chicken and duck were designed. When optimised, the assay could detect pork, chicken and duck down to 0.1% (w/w) in meat mixtures even when the meat sample had undergone freezing, heating and autoclaving. The assay was evaluated by testing 79 commercial beef products collected in local markets, and found that nearly 28% of these contained pork or chicken to varying degrees. The accuracy of the results was verified by repeating the analysis using a standard real-time PCR.
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