Development of Novel Methods for the Concentration and Detection of Foodborne Hepatitis A virus

Abstract

The low amounts of viral particles in contaminated foods make it challenging to develop methods for their detection. Rapid and easy-to-use methods for separating and concentrating viruses from contaminated foods are needed to enhance the efficiency of virus detection. In the present study, iron oxide (Fe3O4) magnetic nanoparticles (MNPs) coated with -NH2 or protamine (designated NMNPs and PMNPs, respectively) were used to concentrate hepatitis A virus (HAV) from different food matrices. Successful coating of -NH2 or protamine was confirmed using Fourier transform infrared spectroscopy (FTIR), zeta potential test, and transmission electron microscopy (TEM). Glycine buffer (0.05 M glycine, 0.14 M NaCl, 0.2% (v/v) Tween 20, pH 9.0) was found to be efficient in eluting HAV from artificially contaminated green onion, strawberry, and mussel. When used for concentrating HAV from the food eluates or milk, PMNPs showed higher sensitivity and repeatability than NMNPs. The detection limit of HAV by real-time RT-PCR was 8.3 plaque-forming unit (PFU)/15 g, 83 PFU/50 g, 8.3 PFU/5 g, and 8.3 PFU/40 mL for green onion, strawberry, mussel, and milk, respectively. Polyethylene glycol (PEG) dialysis method demonstrated similar analytical sensitivity to the PMNP method in concentrating HAV from green onion, mussel, and milk. A bioluminescent real-time reverse transcriptase loop-mediated isothermal amplification (RT-LAMP-BART) technology was employed in the current study to detect HAV concentrated from green onion, strawberry, mussel, and milk by PMNPs. The analytical sensitivity of cDNA-LAMP-BART assay was 8.3 PFU/15 g, 83 PFU/50 g, 8.3 PFU/5 g, and 8.3 PFU/40 mL for green onion, strawberry, mussel, and milk, respectively. These results were comparable with those from real-time RT-PCR. Qβ replicase reaction assay was also investigated in the present study for HAV detection. It was found that the method was not applicable in detecting foodborne virus due to the strong background signal in non-template reactions, which gave false positive results.