In order to establish a real-time recombinase aided amplification (Rt-RAA) method to rapidly detect dog-derived ingredients, three pairs of primers and one exonuclease (exo) probe were designed targeting the conserved region of the cytochrome B (cytb) gene. The designed primer combinations and probe concentration were optimized. The specificity and stability of the method were evaluated by cross-reactivity and repeatability experiments. The sensitivity of the Rt-RAA method and its results for simulated mixed samples and commercial samples were compared with those of the national standard method. The results showed that the Rt-RAA method had strong specificity and good stability and could detect the target sequence in less than 30 min. The detection limit for dog-derived ingredients was 0.2%, which was significantly better than that of real-time fluorescence polymerase chain reaction (PCR). The results of the Rt-RAA method for simulated mixed samples and commercial samples were completely consistent with those of the national standard method. Therefore, Rt-RAA is suitable for the detection of dog-derived ingredients, providing a fast and accurate new method for the identification of meat-derived ingredients.

In this study, real-time recombinase-aided amplification (Rt-RAA) was used for the synchronous detection of rats-and mice-derived components in meat products. Primers and probes targeting the conserved sequences of the COX3 gene in rats and those of the 16S rRNA gene in mice were designed. The reaction parameters were optimized to establish the optimal reaction conditions for duplex Rt-RAA detection. Furthermore, the specificity, sensitivity and stability of the established duplex Rt-RAA method were evaluated, and it was applied to simulated commercial meat products. The results showed that this method had strong specificity and stability. The detection limits for rats- and mice-derived components were 0.5% and 0.2%, respectively. The detection limits for their synchronous detection were both 0.5%, and the detection limits for synchronous detection of rats- and mice-derived components mixed in simulated commercial meat samples were still as low as 0.5%. Using duplex Rt-RAA and real-time fluorescence quantitative polymerase chain reaction (real-time PCR), highly consistent results were obtained for commercially available samples and simulated samples. Therefore, the duplex Rt-RAA method is suitable for the detection of rats- and mice- derived components in meat products, providing a fast, efficient, and accurate new method for the detection of meat product adulteration.