Rapid and simultaneous detection of Salmonella, Shigella, and Staphylococcus aureus in fresh pork using a multiplex real-time PCR assay based on immunomagnetic separation

- This method has low LOD (<10 cfu/g) for Salmonella, Shigella, and Staphylococcus aureus.
- This method has high sensitivity, specificity, and accuracy for these three pathogens.
- The detection time with the new method is ∼10% that of conventional ISO methods.

The incidence of foodborne infections caused by Salmonella sp., Shigella sp. and Staphylococcus (S.) aureus in fresh pork is increasing each year, which poses a great potential threat to public health. In this study, a rapid and simultaneous detection for these three pathogens from fresh pork samples was developed by combining immunomagnetic separation (IMS) with multiplex real-time PCR (RT-PCR). Magnetic beads coated with specific antibodies were used to capture and purify the pathogens from 250 mL matrix prepared by both spiked and commercial samples, followed by DNA extraction. Then, multiplex RT-PCR was applied with three sets of specific primers and probes. The limit of detections were evaluated in 67 spiked pork samples and were 2.0 CFU/g for Salmonella, 6.8 CFU/g for Shigella, and 9.6 CFU/g for S. aureus. The sensitivity, specificity, and accuracy of IMS-multiplex RT-PCR method were 99.2%, 100%, and 99.5%, respectively. One hundred fifty-one samples were tested using the IMS-multiplex RT-PCR and culture methods, and a comparison of the results showed that the former was a potentially reliable method for rapid and effective detection of Salmonella sp., Shigella sp., and S. aureus in fresh pork.