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

摘要

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 105 colony-forming unit (CFU)/mL of V. parahaemolyticus using 2.5 micro g 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 1.4x102 CFU/mL in pure culture and was unaffected by the presence of 108 CFU/mL of competing microflora. When applied in spiked oysters, the sensitivity was found to be 1.9x103 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.