Rapid detection of Vibrio parahaemolyticus in raw oysters using immunomagnetic separation combined with loop-mediated isothermal amplification.
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 2.5μg of IMNPs within 30min. 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 6-8h, the limit of detectability could be improved to 1.9 to 0.19CFU/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.
Food Safety Testing Center, Beijing Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China, Beijing 100026, China. Electronic address: zengj@bjciq.gov.cn.
Jing
J
Zeng
Food Safety Testing Center, Beijing Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China, Beijing 100026, China.
Haiyan
H
Wei
Food Safety Testing Center, Beijing Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China, Beijing 100026, China; Beijing University of Agriculture, Beijing 102206, China.
Lei
L
Zhang
National Center for Nanoscience and Technology, Beijing 100190, China.
Xuefeng
X
Liu
Beijing University of Agriculture, Beijing 102206, China.
Haiyu
H
Zhang
Food Safety Testing Center, Beijing Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China, Beijing 100026, China; Beijing University of Agriculture, Beijing 102206, China.
Jinxia
J
Cheng
Food Safety Testing Center, Beijing Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China, Beijing 100026, China.
Dan
D
Ma
Food Safety Testing Center, Beijing Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China, Beijing 100026, China.
Ximeng
X
Zhang
Food Safety Testing Center, Beijing Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China, Beijing 100026, China.
Pubo
P
Fu
Food Safety Testing Center, Beijing Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China, Beijing 100026, China.
Li
L
Liu
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D005516:Food Microbiology / Q000379:methods*; D018189:Immunomagnetic Separation; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques; D010125:Ostreidae / Q000382:microbiology*; D013997:Time Factors; D014736:Vibrio parahaemolyticus / Q000235:genetics* / Q000302:isolation & purification
D001426:Bacterial Proteins
Immunomagnetic separation; Loop-mediated isothermal amplification (LAMP); Nanoparticle; Oyster; Vibrio parahaemolyticus