A Novel SDS Rinse and Immunomagnetic Beads Separation Combined with Real-Time Loop-Mediated Isothermal Amplification for Rapid and Sensitive Detection of Salmonella in Ready-to-Eat Duck Meat

Abstract

In this study, a real-time loop-mediated isothermal amplification (Rti-LAMP) combined with immunomagnetic beads separation (IMS) assay and SDS rinse was developed for the rapid and sensitive detection of Salmonella in ready-to-eat (RTE) meat without prior enrichment. Polyclonal antibody and specific immunomagnetic beads (IMBs) for Salmonella flagellum were prepared. The capture efficiency was more than 95% by adding 50 μL IMBs into 1 mL of Salmonella suspension for absorption at 37 °C for 20 min. Then, Gly-HCl was the key to the effective eluting and separating of bacteria from IMB-bacterial complexes. The eluted bacteria were resuspended with 1× TZ lysing solution and boiled at 99.5 °C for 10 min, as DNA template of Rti-LAMP assay. The development of IMS-Rti-LAMP assay could specifically detect Salmonella as low as 5 CFU/mL in 0.85% saline solution. The lowest level of Salmonella DNA consistently detected by the IMS-Rti-LAMP assay was that from 75 CFU per 25 g (3 CFU/g) of RTE duck neck meat. The whole assay could be completed in 3 h. In addition, the IMS-Rti-LAMP assay was used to detect 22 RTE duck meat samples from local market, using plate culture as control. The result showed that four samples were positive for Salmonella by IMS-Rti-LAMP assay; three of them were also positive by plate culture method. These results showed that the development of IMS-Rti-LAMP combined with SDS rinsing was a rapid and sensitive method for the detection of Salmonella in RTE duck meat without enrichment.