Detecting Thermal Inactivation of Human Norovirus on Spinach Using Propidium or Ethidium Monoazide Combined with Real-time Quantitative Reverse Transcription-polymerase Chain Reaction

Introduction: Human norovirus (HuNoV), as a serious viral agent, is the main cause of acute gastroenteritis diseases worldwide. An obstacle in HuNoV research concerns the inability of the virus to be cultivated in all environments. Propidium monoazide (PMA) and ethidium monoazide (EMA) can be applied, as a pretreatment, prior to real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to distinguish between live and dead cells.

Purpose: In Korea, spinach is mostly cooked by blanching, followed by seasoning with soy sauce and other condiments to manufacture muchim. Therefore, the present study aimed to evaluate the inhibitory efficacy of heat against HuNoV in suspension (6 to 85°C, for one minute) and on spinach (6 to 95°C, for two minutes) by applying PMA- or EMA-pretreatment combined with qRT-PCR (PMA/qRT-PCR or EMA/qRT-PCR).

Methods: HuNoV stock samples, in 500 μl aliquots (approximately seven log copy number/μl), were heated for one minute in a water bath set at 65, 75, or 85°C. Spinach samples, which were inoculated with 1,500 μl of HuNoV suspension, were heated for two minutes in a water bath at 65, 75, 85, and 95°C. The inactivation effects of thermal treatment against human norovirus (HuNoV) in suspension and on spinach was evaluated by using qRT-PCR, EMA/qRT-PCR, and PMA/qRT-PCR.

Results: Total titers of non-dye treated, EMA-treated, or PMA-treated HuNoV in suspension were significantly (P<0.05) reduced to 0.22 to 0.77, 0.42 to 2.42, and 0.54 to 2.96 log copy number/μl, respectively, after thermal exposure at 65 to 85°C for one minute. HuNoV titers on spinach were, also, significantly (P<0.05) reduced to 0.27 to 1.01, 0.34 to 2.39, and 0.82 to 2.59 log copy number/μl in qRT-PCR, EMA/qRT-PCR, and PMA/qRT-PCR, respectively, after treatment at 65 to 85°C for two minutes.

Significance: HuNoV, in suspension or on spinach, treated with PMA or EMA exhibited a greater decrease compared with nondye treatments at all heating temperatures.