Rapid detection of Vibrio parahaemolyticus in raw oysters using immunomagnetic separation combined with loop-mediated isothermal amplification

Abstract The objective of this study was to develop a method that combined nanoparticle-based immunomagnetic separation (IMS) with real-time loop-mediated isothermal amplification (LAMP) for the rapid detection of Vibrio parahaemolyticus . Magnetic nanoparticles were functionalized with monoclonal antibodies that were produced against flagella from V . parahaemolyticus to capture and separate the target cells from raw oysters. After optimization, the immunomagnetic nanoparticles (IMNPs) presented a capture efficiency of 87.3% for 10 5 colony-forming unit (CFU)/mL of V . parahaemolyticus using 2.5 μg of IMNPs within 30 min. Although a very low level of non-specific binding was seen among 8 non- V . parahaemolyticus Vibrio spp. and 5 non- Vibrio strains, the IMS–LAMP method identified 133 V . parahaemolyticus strains correctly without the amplification from 54 other strains. The detection limit was about 1.4 × 10 2  CFU/mL in pure culture and was unaffected by the presence of 10 8  CFU/mL of competing microflora. When applied in spiked oysters, the sensitivity was found to be 1.9 × 10 3  CFU/g without enrichment. After enrichment for 6–8 h, the limit of detectability could be improved to 1.9 to 0.19 CFU/g. Hence, the IMS–LAMP assay provided a rapid, simple, and cost-effective method for total V . parahaemolyticus detection. This method will have important implications in the rapid detection of contaminated food in the early stage before distribution.