Thermal inactivation of human norovirus on spinach using propidium or ethidium monoazide combined with real-time quantitative reverse transcription-polymerase chain reaction

Abstract

The present study investigated the inactivation effects of thermal treatment against human norovirus (HuNoV) in suspension and on spinach by using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), ethidium monoazide combined with qRT-PCR (EMA/qRT-PCR), and propidium monoazide combined with qRT-PCR (PMA/qRT-PCR). Total titers of non-dye treated, EMA-treated, or PMA-treated HuNoV in suspension were significantly (P < 0.05) reduced to 0.22–0.77, 0.42–2.42, and 0.54–2.96 log10 copy number/μL, respectively, after thermal exposure at 65–85 °C for 1 min. HuNoV titers on spinach were significantly (P < 0.05) reduced to 0.27–1.01, 0.34–2.39, and 0.82–2.59 log10 copy number/μL in qRT-PCR, EMA/qRT-PCR, and PMA/qRT-PCR, respectively, after treatment at 65–85 °C for 2 min. Non-dye treated HuNoV on spinach exhibited a decrease of <1.5 log10 copy number/μL, whereas EMA-treated or PMA-treated HuNoV on spinach was not detected after treatment at 95 °C for 2 min. Specifically, HuNoV with two dye treatments exhibited a greater decrease compared with non-dye treatment at all heating temperatures in suspension, as well as on spinach. The difference in reduction values between non-dye treatment and dye-pretreatments increased gradually along with the stepwise increase in temperature. Based on these results, qRT-PCR combined with EMA or PMA could be regarded as a more useful method as compared with qRT-PCR alone to discriminate the viability of thermally treated HuNoV. Furthermore, despite a similar decreasing trend observed following both dye-pretreatments, HuNoV titer decreased slightly more with PMA treatment than with EMA treatment under all thermal conditions.