Development of an Extraction Method to Detect Hepatitis A Virus, Hepatitis E Virus, and Noroviruses in Fish Products

Viruses are a leading cause of foodborne disease worldwide. Hepatitis viruses (hepatitis A (HAV) and hepatitis E (HEV)) and human norovirus are recognized as the main viruses of public health concern in food hygiene. ISO 15216 approved procedures are not validated for detection of HAV and human norovirus in foodstuffs, such as fishes, leading to an inability to ensure the safety of these products. This study aimed to provide a rapid and sensitive method for detecting these targets in fish products. An existing method that includes proteinase K treatment was selected for further validation using artificially contaminated fish products, according to the recent international standard ISO 16140-4. Recovery efficiencies in pure RNA extracts of viruses ranged from 0.2% to 66.2% for HAV, 4.0% to 100.0% for HEV, 2.2% to 100.0% for norovirus GI, and 0.2% to 12.5% for norovirus GII. LOD₅₀ values were between 144 and 8.4 × 10⁴ genome copies/g for HAV and HEV, and 10⁴ and 2.0 × 10³ copies/g for norovirus GI and norovirus GII, respectively. LOD₉₅ values were between 3.2 × 10³ and 3.6 × 10⁵ genome copies/g for HAV and HEV, and between 8.8 × 10³ and 4.4 × 10⁴ genome copies/g for norovirus GI and norovirus GII, respectively. The method developed here was successfully validated in various fish products and can be applied for routine diagnostic needs.