PMAxx-RT-qPCR to Determine Human Norovirus Inactivation Following High-Pressure Processing of Oysters.

Norovirus is the leading cause of viral gastroenteritis globally. While person-to-person transmission is most commonly reported route of infection, human norovirus is frequently associated with foodborne transmission, including through consumption of contaminated bivalve molluscan shellfish. Reverse transcription (RT)-qPCR is most commonly used method for detecting human norovirus detection in foods, but does not inform on its infectivity, posing challenges for assessing intervention strategies aimed at risk elimination. In this study, RT-qPCR was used in conjunction with a derivative of the photoreactive DNA binding dye propidium monoazide (PMAxx™) (PMAxx-RT-qPCR) to evaluate the viral capsid integrity of norovirus genogroup I and II (GI and GII) in shellfish following high pressure processing (HPP). Norovirus GI.3 and GII.4 bioaccumulated oysters were subjected to HPP at pressures of 300 and 450 MPa at 15 °C, and 300, 450 and 600 MPa at 20 °C. Samples were analysed using both RT-qPCR and PMAxx-RT-qPCR. For each sample, norovirus concentration (genome copies/g digestive tissue) determined by RT-qPCR was divided by the PMAxx-RT-qPCR concentration, giving the relative non-intact (RNI) ratio. The RNI ratio values relate to the amount of non-intact (non-infectious) viruses compared to fully intact (possible infectious) viruses. Our findings revealed an increasing RNI ratio value, indicating decreasing virus integrity, with increasing pressure and decreasing pressure. At 300 MPa, for norovirus GI, the median [95% confidence interval, CI] RNI ratio values were 2.6 [1.9, 3.0] at 15 °C compared to 1.1 [0.9, 1.8] at 20 °C. At 450 MPa, the RNI ratio values were 5.5 [2.9, 7.0] at 15 °C compared to 1.3 [1.0, 1.6] at 20 °C. At 600 MPa, the RNI ratio value was 5.1 [2.9, 13.4] at 20 °C. For norovirus GII, RT-qPCR and PMAxx-RT-qPCR detections were significantly reduced at 450 and 600 MPa at both 15 °C and 20 °C, with the median [95% CI] RNI ratio value at 300 MPa being 1.1 [0.8, 1.6]. Following HPP treatment, the use of PMAxx-RT-qPCR enables the selective detection of intact and potential infectious norovirus, enhancing our understanding of the inactivation profiles and supporting the development of more effective risk assessment strategies.

Identifying Suitable Listeria innocua Strains as Surrogates for Listeria monocytogenes for Horticultural Products.

A laboratory-based study testing 9 Listeria innocua strains independently and a cocktail of 11 Listeria monocytogenes strains was carried out. The aim was to identify suitable L. innocua strain(s) to model L. monocytogenes in inactivation experiments. Three separate inactivation procedures and a hurdle combination of the three were employed: thermal inactivation (55°C), UV-C irradiation (245 nm), and chemical sanitizer (TsunamiTM 100, a mixture of acetic acid, peroxyacetic acid, and hydrogen peroxide). The responses were strain dependent in the case of L. innocua with different strains responding differently to different regimes and L. innocua isolates generally responded differently to the L. monocytogenes cocktail. In the thermal inactivation treatment, inactivation of all strains including the L. monocytogenes cocktail plateaued after 120 min. In the case of chemical sanitizer, inactivation could be achieved at concentrations of 10 and 20 ppm with inactivation increasing with contact time up to 8 min, beyond which there was no significant benefit. All L. innocua strains except PFR16D08 were more sensitive than the L. monocytogenes cocktail to the hurdle treatment. PFR16D08 almost matched the resistance of the L. monocytogenes cocktail but was much more resistant to the individual treatments. A cocktail of two L. innocua strains (PFR 05A07 and PFR 05A10) had the closest responses to the hurdle treatment to those of the L. monocytogenes cocktail and is therefore recommended for hurdle experiments.