2.4. Viability dye treatmentsOne-hu

2.4. Viability dye treatments

One-hundred microliters of lettuce wash water or food concentrates was inoculated with infectious HAV and thermally inactivated (5 min at 99 °C) HAV suspensions (at concentrations of ca. 6 × 104, 6 × 103 and 6 × 102 TCID50), and added to PMA 50 μM and EMA 20 μM, with or without surfactants. All the experiments were performed in DNA LoBind 1.5 ml tubes (Eppendorf) in triplicate. After the addition of the viability dye, incubation in the dark at room temperature was performed for 10 min at 150 rpm to allow reagent penetration. Thereafter, samples were exposed to light for 15 min using a photo-activation system (Led-Active Blue, Geniul). After photo-induced cross-linking, RNA was extracted using the NucleoSpin® RNA virus kit (Macherey-Nagel GmbH & Co.) according to the manufacturer's instructions. Three types of controls were always included in the experiments; infectious viruses treated with viability dyes and infectious and thermally inactivated viruses without viability dye treatment.